DNA encoding the human serine protease EOS

ABSTRACT

Here we describe the molecular identification of a cDNA encoding a novel serine protease we have termed protease EOS. The deduced amino acid sequence, and it alignment with other well-characterized serine proteases indicates that it is a member of the S1 serine protease family. We have found that the protease EOS mRNA is expressed in platelets and leukocytes and more specifically eosinophils. Although this protease is abundantly expressed in ovary, retina and stomach, where it may perform important functions, its expression in platelets and certain cells of the immune system suggests that it may play roles in thrombosis and in the immune process. Enzymatically active protease EOS is amenable to further biochemical analyses for the identification of physiological substrates and specific modulators

BACKGROUND OF THE INVENTION

[0001] Members of the trypsin/chymotrypsin-like (S1) serine proteasefamily play pivotal roles in a multitude of diverse physiologicalprocesses, including digestive processes and regulatory amplificationcascades through the proteolytic activation of inactive zymogenprecursors. In many instances protease substrates within these cascadesare themselves the inactive form, or zymogen, of a “downstream” serineprotease. Well-known examples of serine protease-mediated regulationinclude blood coagulation, (Davie, et al (1991). Biochemistry30:10363-70), kinin formation (Proud and Kaplan (1988). Ann Rev Immunol6:49-83) and the complement system (Reid and Porter (1981). Ann RevBiochemistry 50:433-464). Although these proteolytic pathways have beenknown for sometime, it is likely that the discovery of novel serineprotease genes and their products will enhance our understanding ofregulation within these existing cascades, and lead to the elucidationof entirely novel protease networks.

[0002] Differentiated blood cells express an assortment of proteasesthat are likely to play specific roles in various pathological states.Although granzymes from cytotoxic T cells and natural killer (NK) cells(Smyth et al. (1996). J. Leukocyte Biol. 60:555-562), elastase andcollagenases from neutrophils (Simon (1993). Agents Actions Suppl.42:27-37) and chymase and tryptase from mast cells (Caughey (1995).Clin. Allergy Immunol. 6:305-29; Katunuma and Kido (1988). J. Cell.Biochem. 38:291-301) are currently under investigation, their roles inpathophysiological processes are only now being elucidated. In contrast,the proteases from eosinophils have not been characterized and are onlycurrently being molecularly identified. Understanding the physiologicalroles these eosinophil proteases play will lead to a betterunderstanding of eosinophil function in health and diseased states(Abu-Ghazaleh et al. (1992). Immunol. Ser. 57:137-67; Gleich (1996).Allergol. Int. 45:35-44; Gleich et al. (1993). Annu. Rev. Med.44:85-101). Proteases are used in non-natural environments for variouscommercial purposes including laundry detergents, food processing,fabric processing, and skin care products. In laundry detergents, theprotease is employed to break down organic, poorly soluble compounds tomore soluble forms that can be more easily dissolved in detergent andwater. In this capacity the protease acts as a “stain remover.” Examplesof food processing include tenderizing meats and producing cheese.Proteases are used in fabric processing, for example, to treat wool inorder prevent fabric shrinkage. Proteases may be included in skin careproducts to remove scales on the skin surface that build up due to animbalance in the rate of desquamation. Common proteases used in some ofthese applications are derived from prokaryotic or eukaryotic cells thatare easily grown for industrial manufacture of their enzymes, forexample a common species used is Bacillus as described in U.S. Pat. No.5,217,878. Alternatively, U.S. Pat. No. 5,278,062 describes serineproteases isolated from a fungus, Tritirachium album, for use in laundrydetergent compositions. Unfortunately use of some proteases is limitedby their potential to cause allergic reactions in sensitive individualsor by reduced efficiency when used in a non-natural environment. It isanticipated that protease proteins derived from non-human sources wouldbe more likely to induce an immune response in a sensitive individual.Because of these limitations, there is a need for alternative proteasesthat are less immunogenic to sensitive individuals and/or providesefficient proteolytic activity in a non-natural environment. The adventof recombinant technology allows expression of any species' proteins ina host suitable for industrial manufacture.

SUMMARY OF THE INVENTION

[0003] Here we describe the molecular identification of a cDNA encodinga novel serine protease we have termed protease EOS. The protease EOScDNA sequence predicts a preproEOS polypeptide of 284 amino acids, andits alignment with other well-characterized serine proteases clearlyindicates that it is a member of the S1 serine protease family.

[0004] Enzymatically active protease EOS is amenable to furtherbiochemical analyses for the identification of physiological substratesand specific modulators. Modulators identified in the chromogenic assaydisclosed herein are potentially useful as therapeutic agents in thetreatment of diseases associated with platelet function or elevatedeosinophil counts such as in, but not limited to, bronchial asthma andcomplications arising from hypereosinophilia. In addition, expression ofprotease EOS in the ovary, retina and stomach suggests that modulatorsof protease EOS function could be used to treat disorders effectingthese tissues. Purified protease EOS can be manufactured as a componentfor use in commercial products including laundry detergents,stain-removing solutions, and skin care products.

[0005] The recombinant DNA molecules coding for EOS, and portionsthereof, are useful for isolating homologues of the DNA molecules,identifying and isolating genomic equivalents of the DNA molecules, andidentifying, detecting or isolating mutant forms of the DNA molecules

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1—The nucleotide (SEQ. ID. NO.::1) and amino acid sequence(SEQ. ID. NO.:7) of the novel protease EOS cDNA is shown.

[0007]FIG. 2—The phylogenetic tree of the protease EOS amino acidsequence relative to other S1 serine proteases is shown.

[0008]FIG. 3—PCR-based tissue distribution indicates that the proteaseEOS mRNA is restricted. Autoradiograms of gels are shown with theposition of the EOS specific PCR product (EOS), as detected by thehybridization of a labeled nested probe, which was resolved followingelectrophoresis from the free probe (F.P.). The cDNA libraries oftissues and cell lines analyzed are as indicated.

[0009]FIG. 4—The nucleotide and amino acid sequences of the protease EOScatalytic domain in the zymogen activation construct is shown.

[0010]FIG. 5—Polyacrylamide gel and Western blot analyses of therecombinant protease PFEK-protease EOS-HA6XHIS. Shown is thepolyacrylamide gel containing samples of the novel serine proteasePFEK-protease EOS-HA6XHIS stained with Coomassie Brilliant Blue(Leftmost 1, 2.). The relative molecular masses are indicated by thepositions of protein standards (M) . In the indicated lanes, thepurified zymogen was either untreated (−) or digested with EK (+) whichwas used to cleave and activate the zymogen into its active form.Western blot of the gel, probed with the anti-FLAG MoAb M2, is alsoshown (rightmost 1). This demonstrates the quantitative cleavage of theexpressed and purified zymogen to generate the processed and activatedprotease.

[0011]FIG. 6—Functional amidolytic activities of the recombinantprotease EOS-HA6XHIS expressed, purified and activated from theactivation construct were determined using chromogenic substrates.

DETAILED DESCRIPTION

[0012] Definitions:

[0013] The term “protein domain” as used herein refers to a region of aprotein that can fold into a stable three-dimensional structureindependent to the rest of the protein. This structure may maintain aspecific function associated with the domain's function within theprotein including enzymatic activity, creation of a recognition motiffor another molecule, or provide necessary structural components for aprotein to exist in a particular environment. Protein domains areusually evolutionarily conserved regions of proteins, both within aprotein superfamily and within other protein superfamilies that performsimilar functions. The term “protein superfamily” as used herein refersto proteins whose evolutionary relationship may not be entirelyestablished or may be distant by accepted phylogenetic standards, butshow similar three dimensional structure or display unique consensus ofcritical amino acids. The term “protein family” as used herein refers toproteins whose evolutionary relationship has been established byaccepted phylogenic standards.

[0014] The term “fusion protein” as used herein refers to proteinconstructs that are the result of combining multiple protein domains orlinker regions for the purpose of gaining function of the combinedfunctions of the domains or linker regions. This is most oftenaccomplished by molecular cloning of the nucleotide sequences to resultin the creation of a new polynucleotide sequence that codes for thedesired protein. Alternatively, creation of a fusion protein may beaccomplished by chemically joining two proteins together.

[0015] The term “linker region” or “linker domain” or similar suchdescriptive terms as used herein refers to stretches of polynucleotideor polypeptide sequence that are used in the construction of a cloningvector or fusion protein. Functions of a linker region can includeintroduction of cloning sites into the nucleotide sequence, introductionof a flexible component or space-creating region between two proteindomains, or creation of an affinity tag for specific moleculeinteraction. A linker region may be introduced into a fusion proteinwithout a specific purpose, but results from choices made duringcloning.

[0016] The term “pre-sequence” as used herein refers to a nucleotidesequence that encodes a secretion signal amino acid sequence. A widevariety of such secretion signal sequences are known to those skilled inthe art, and are suitable for use in the present invention. Examples ofsuitable pre-sequences include, but are not limited to, prolactinFLAG,trypsinogen, and chymoFLAG.

[0017] The term “pro-sequence” as used herein refers to a nucleotidesequence that encodes a cleavage site for a restriction protease. A widevariety of cleavage sites for restriction proteases are known to thoseskilled in the art, and are suitable for use in the present invention.Examples of suitable pro-sequences include, but are not limited to, EK,FXa, and thrombin.

[0018] The term “cloning site” or “polycloning site” as used hereinrefers to a region of the nucleotide sequence contained within a cloningvector or engineered within a fusion protein that has one or moreavailable restriction endonuclease consensus sequences. The use of acorrectly chosen restriction endonuclease results in the ability toisolate a desired nucleotide sequence that codes for an in-framesequence relative to a start codon that yields a desirable proteinproduct after transcription and translation. These nucleotide sequencescan then be introduced into other cloning vectors, used create novelfusion proteins, or used to introduce specific site-directed mutations.It is well known by those in the art that cloning sites can beengineered at a desired location by silent mutations, conservedmutation, or introduction of a linker region that contains desiredrestriction enzyme consensus sequences. It is also well known by thosein the art that the precise location of a cloning site can be flexibleso long as the desired function of the protein or fragment thereof beingcloned is maintained.

[0019] The term “tag” as used herein refers to a nucleotide sequencethat encodes an amino acid sequence that facilitates isolation,purification or detection of a fusion protein containing the tag. A widevariety of such tags are known to those skilled in the art, and aresuitable for use in the present invention. Suitable tags include, butare not limited to, HA-tag, His-tag, biotin, avidin, and antibodybinding sites.

[0020] As used herein, “expression vectors” are defined herein as DNAsequences that are required for the transcription of cloned copies ofgenes and the translation of their mRNAs in an appropriate host. Suchvectors can be used to express eukaryotic genes in a variety of hostssuch as bacteria including E. coli, blue-green algae, plant cells,insect cells, fungal cells including yeast cells, and animal cells.

[0021] The term “catalytic domain cassette” as used herein refers to anucleotide sequence that encodes an amino acid sequence encoding atleast the catalytic domain of the serine protease of interest. A widevariety of protease catalytic domains may be inserted into theexpression vectors of the present invention, including those presentlyknown to those skilled in the art, as well as those not yet having anisolated nucleotide sequence encodes it, once the nucleotide sequence isisolated.

[0022] As used herein, a “functional derivative” of the nucleotidesequence, vector, or polypeptide possesses a biological activity (eitherfunctional or structural) that is substantially similar to theproperties described herein. The term “functional derivatives” isintended to include the “fragments,” “variants,” “degenerate variants,”“analogs” and “homologues” of the nucleotide sequence, vector, orpolypeptide. The term “fragment” is meant to refer to any nucleotidesequence, vector, or polypeptide subset of the modules described as preand pro sequences used for the activation of expressed zymogenprecursors. The term “variant” is meant to refer to a nucleotide oramino acid sequence that is substantially similar in structure andfunction to either the entire nucleic acid sequence or encoded proteinor to a fragment thereof. A nucleic acid or amino acid sequence is“substantially similar” to another if both molecules have similarstructural characteristics or if both molecules possess similarbiological properties. Therefore, if the two molecules possesssubstantially similar activity, they are considered to be variants evenif the structure of one of the molecules is not found in the other oreven if the two amino acid sequences are not identical. The term“analog” refers to a protein molecule that is substantially similar infunction to another related protein.

[0023] Herein we describe a serine protease isolated from eosinophilcells termed EOS. The protease EOS deduced amino acid sequence is mostsimilar to the cloned serine proteases prostasin (Yu et al. (1996).Genomics 32:334-40) and tryptase (Miller et al. (1990). J. Clin. Invest.86:864-700). Tryptase, which is produced abundantly in mast cells, hasbeen implicated in asthmatic inflammation and obstruction (Johnson etal. (1997). Eur. Respir. J. 10:38-43). Additional homology searches ofthe Genbank database with the protease EOS nucleotide sequence revealedhomology with non-contiguous regions of the human cosmid clone (407D8,Genbank accession # AC005570), which maps to chromosome 16p13.3.Assembly of a continuous nucleic acid sequence from the proposedintron/exon junctions described in the Genbank accession # AC005570annotation produces a nucleic acid sequence that is shorter and alsonon-contiguous, and thus substantially different from, protease EOS ofthe present invention.

[0024] Thus, it is likely that the exons delineated in the Genbankaccession # AC005570 annotation are incorrect. Therefore, protease EOSof the present invention represents a previously undescribed protease.The use of the previously undescribed sequence of the present inventionindicates that chromosome 16p13.3 is the correct the position of theprotease EOS gene. Interestingly, the gene encoding prostasin has beenlocalized on chromosome 16p11.2 (Yu et al. (1996). Genomics 32:334-40)while several tryptase genes are even clustered within the samechromosome 16p13.3 genomic interval (Pallaoro et al. (1999). J. Biol.Chem. 274:3355-3362), and are consequently co-localized with the genefor protease EOS. Recent genetic data links determinants ofsusceptibility to asthma on chromosome 16 in some populations (Danielset al. (1996). Nature (London) 383:247-253). Eosinophilia, a conditioncharacterized by elevated circulating eosinophils, is associated withnumerous allergic states including bronchial asthma (Gleich (1996).Allergol. Int. 45:35-44). Therefore protease EOS, or manipulation ofthis enzyme by chemical modulators, may be useful for treatment ofimmune processes involving eosinophils, with asthma being one importantexample.

[0025] Proteases are used in non-natural environments for variouscommercial purposes including laundry detergents, food processing,fabric processing, and skin care products. In laundry detergents, theprotease is employed to break down organic, poorly soluble compounds tomore soluble forms that can be more easily dissolved in detergent andwater. In this capacity the protease acts as a “stain remover.” Examplesof food processing include tenderizing meats and producing cheese.Proteases are used in fabric processing, for example, to treat wool inorder prevent fabric shrinkage. Proteases may be included in skin careproducts to remove scales on the skin surface that build up due to animbalance in the rate of desquamation. Unfortunately use of someproteases is limited by their potential to cause allergic reactions insensitive individuals or by reduced efficiency when used in anon-natural environment. Because of these limitations, there is a needfor alternative proteases that are less immunogenic to sensitiveindividuals and/or provides efficient proteolytic activity in anon-natural environment. Because protease EOS is derived from a humanhost, it is less likely to induce an allergic reaction in sensitiveindividuals, and therefore protease EOS may also be useful forformulation of compositions for laundry detergents and skin careproducts.

[0026] The present invention relates to DNA encoding the serine proteaseEOS that was identified from an eosinophil library, constructed usingpoly A RNA isolated from pooled diseased eosinophils obtained fromallergic asthmatic individuals. The protease EOS as used herein, refersto the encoded protein product which can specifically function as aprotease.

[0027] The complete amino acid sequence of protease EOS was notpreviously known, nor was the complete nucleotide sequence encodingprotease EOS known. This is the first reported cloning of a full lengthDNA molecule encoding protease EOS. Based on mRNA distribution, it ispredicted that a restricted number of tissues and cell types willcontain the described protease. Vertebrate cells capable of producingprotease EOS include, but are not limited to eosinophils isolated fromblood. Other tissue types may be human spleen, retina, spinal cord andovary.

[0028] Other cells and cell lines may also be suitable for use toisolate the protease EOS cDNA. Selection of suitable cells may be doneby screening for protease EOS proteolytic activity in conditioned cellmedia. Cell types that possess EOS proteolytic activity in this assaymay be suitable for the isolation of the protease EOS DNA or mRNA.

[0029] Any of a variety of procedures known in the art may be used tomolecularly clone protease EOS DNA. These methods include, but are notlimited to, direct functional expression of protease genes following theconstruction of a protease EOS-containing cDNA library in an appropriateexpression vector system. Another method is to screen proteaseEOS-containing cDNA library constructed in a bacteriophage or plasmidshuttle vector with a labeled oligonucleotide probe designed from theamino acid sequence of the protease EOS DNA. An additional methodconsists of screening a protease EOS-containing cDNA library constructedin a bacteriophage or plasmid shuttle vector with a partial cDNAencoding the protease EOS protein. This partial cDNA is obtained by thespecific polymerase chain reaction (PCR) amplification of protease EOSDNA fragments through the design of degenerate oligonucleotide primersfrom the amino acid sequence of the purified protease EOS protein.Expressed sequence tags (EST)s, identified through homology searching ofnucleic acid databases (Altschul et al. (1990). J. Mol. Biol.215:403-10; Pearson and Lipman (1988). Proc. Natl. Acad. Sci. U.S.A.85:2444-8), are also available for this purpose. This particularprotease is a member of a multigene family containing highly conservedresidues and motifs. Thus, cDNA library screening under reducedstringency to identify related but non-identical homologous cDNAs ispossible. More recently, direct PCR using degenerate oligonucleotides ofcDNA reverse transcribed from RNA of a given cell type, has been afruitful approach to isolate novel related cDNAs of interest.Alternatively, the full-length cDNA sequence once published, may beobtained by the specific PCR amplification, through the design ofmatching oligonucleotide primers flanking the entire coding sequence.

[0030] Another method is to isolate RNA from protease EOS-producingcells and translate the RNA into protein via an in vitro or an in vivotranslation system. The translation of the RNA into a protein willresult in the production of at least a portion of the protease EOSprotein that can be identified by, for example, immunological reactivitywith an anti-protease EOS antibody. Should the entire catalytic domainbe translated, functional proteolytic activity of the EOS protein couldconceivably be used to identify RNA fractions containing the proteaseEOS mRNA. In this method, pools of RNA isolated from proteaseEOS-producing cells can be analyzed for the presence of an RNA thatencodes at least a portion of the EOS protein. Further fractionation ofthe RNA pool can be done to purify the protease EOS RNA fromnon-protease EOS RNA. The peptide or protein produced by this method maybe analyzed to provide amino acid sequences, which in turn may be usedto provide primers for production of protease EOS cDNA. Similarly, RNAused for translation can be analyzed to provide nucleotide sequences andmay be used to produce probes for the production of the protease EOScDNA. This method is known in the art and can be found in, for example,(Maniatis et al. (1989). 1-1626).

[0031] It is readily apparent to those skilled in the art that othertypes of libraries, as well as libraries constructed from other cells orcell types, may be useful for isolating protease EOS-encoding DNA. Othertypes of libraries include, but are not limited to, cDNA librariesderived from other cells, from non-human organisms, and genomic DNAlibraries that include YAC (yeast artificial chromosome) and cosmidlibraries.

[0032] It is readily apparent to those skilled in the art that suitablecDNA libraries may be prepared from cells or cell lines which have EOSproteolytic activity. The selection of cells or cell lines for use inpreparing a cDNA library to isolate the protease EOS cDNA may be done byfirst measuring cell associated EOS proteolytic activity using themeasurement of protease EOS-associated biological activity or a EOSspecific immunological reactivity.

[0033] Preparation of cDNA libraries can be performed by standardtechniques well known in the art. Well known cDNA library constructiontechniques can be found for example, in (Maniatis et al. (1989).1-1626).

[0034] It is also readily apparent to those skilled in the art that DNAencoding protease EOS may also be isolated from a suitable genomic DNAlibrary. Construction of genomic DNA libraries can be performed bystandard techniques well known in the art. Well known genomic DNAlibrary construction techniques can be found in (Maniatis et al. (1989).1-1626).

[0035] In order to clone the protease EOS gene by the above methods, theamino acid sequence of protease EOS may be necessary. To accomplishthis, the protease EOS protein may be purified and partial amino acidsequence determined by automated sequencers. It is not necessary todetermine the entire amino acid sequence, but the linear sequence of tworegions of 6 to 8 amino acids from the protein is determined for theproduction of primers for PCR amplification of a partial protease EOSDNA fragment. Alternatively, a longer degenerate oligonucleotide probecan be synthesized with a larger consecutive stretch of amino acidsequence determined. This oligonucleotide probe can be labeled and usedto screen a suitable cDNA or genomic library, under the appropriatestringency, to isolate DNA corresponding to protease EOS.

[0036] Once suitable amino acid sequences have been identified, the DNAsequences capable of encoding them are synthesized. Because the geneticcode is degenerate, more than one codon may be used to encode aparticular amino acid, and therefore, the amino acid sequence can beencoded by any of a set of similar DNA oligonucleotides. Only one memberof the set will be identical to the protease EOS sequence, but will becapable of hybridizing to protease EOS DNA even in the presence of DNAoligonucleotides with mismatches. The mismatched DNA oligonucleotidesmay still sufficiently hybridize to the protease EOS DNA to permitidentification and isolation of protease EOS encoding DNA. DNA isolatedby these methods can be used to screen DNA libraries from a variety ofcell types, from invertebrate and vertebrate sources, and to isolatehomologous genes.

[0037] Purified biologically active protease EOS may have severaldifferent physical forms. Protease EOS may exist as a full-lengthnascent or unprocessed polypeptide, or as partially processedpolypeptides or combinations of processed polypeptides. The full-lengthnascent protease EOS polypeptide may be posttranslationally modified byspecific proteolytic cleavage events, which result in the formation offragments of the full-length nascent polypeptide. A fragment, orphysical association of fragments may have the full biological activityassociated with protease EOS however, the degree of protease EOSactivity may vary between individual protease EOS fragments andphysically associated protease EOS polypeptide fragments.

[0038] The cloned protease EOS DNA obtained through the methodsdescribed herein may be recombinantly expressed by molecular cloninginto an expression vector containing a suitable promoter and otherappropriate transcription regulatory elements, and transferred intoprokaryotic or eukaryotic host cells to produce recombinant protease EOSprotein. Techniques for such manipulations are fully described (Maniatiset al. (1989). 1-1626), and are well known in the art.

[0039] Expression vectors are defined herein as DNA sequences that arerequired for the transcription of cloned copies of genes and thetranslation of their mRNAs in an appropriate host. Such vectors can beused to express eukaryotic genes in a variety of hosts such as bacteriaincluding E. coli, blue-green algae, plant cells, insect cells, fungalcells including yeast cells, and animal cells.

[0040] Specifically designed vectors allow the shuttling of DNA betweenhosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungalcells or bacteria-invertebrate cells. An appropriately constructedexpression vector should contain: an origin of replication forautonomous replication in host cells, selectable markers, a limitednumber of useful restriction enzyme sites, a potential for high copynumber, and active promoters. A promoter is defined as a DNA sequencethat directs RNA polymerase to bind to DNA and initiate RNA synthesis. Astrong promoter is one that causes mRNAs to be initiated at highfrequency. Expression vectors may include, but are not limited to,cloning vectors, modified cloning vectors, specifically designedplasmids or viruses.

[0041] A variety of mammalian expression vectors may be used to expressrecombinant protease EOS in mammalian cells. Commercially availablemammalian expression vectors which may be suitable for recombinantprotein expression, include but are not limited to, pCI Neo (Promega,Madison, Wis., Madison Wis.), pMAMneo (Clontech, Palo Alto, Calif.),pcDNA3 (InVitrogen, San Diego, Calif.), pMC1neo (Stratagene, La Jolla,Calif.), pXT1 (Stratagene, La Jolla, Calif.), pSG5 (Stratagene, LaJolla, Calif.), EBO-pSV2-neo (ATCC 37593) pBPV-1 (8-2) (ATCC 37110),pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and 1ZD35 (ATCC37565).

[0042] A variety of bacterial expression vectors may be used to expressrecombinant protease EOS in bacterial cells. Commercially availablebacterial expression vectors which may be suitable for recombinantprotein expression include, but are not limited to pET vectors (Novagen,Inc., Madison Wis.) and pQE vectors (Qiagen, Valencia, Calif.) PGEX(Pharmacia Biotech Inc., Piscataway, N.J.).

[0043] A variety of fungal cell expression vectors may be used toexpress recombinant protease EOS in fungal cells such as yeast.Commercially available fungal cell expression vectors which may besuitable for recombinant protease EOS expression include but are notlimited to pYES2 (InVitrogen, San Diego, Calif.) and Pichia expressionvector (InVitrogen, San Diego, Calif.).

[0044] A variety of insect cell expression systems may be used toexpress recombinant protease EOS in insect cells. Commercially availablebaculovirus transfer vectors which may be suitable for the generation ofa recombinant baculovirus for recombinant protein expression in Sf9cells include but are not limited to pFastBacl (Life Technologies,Gaithersberg, Md.) pAcSG2 (Pharmingen, San Diego, Calif.) pBlueBacII(InVitrogen, San Diego, Calif.). In addition, a class of insect cellvectors that permit the expression of recombinant proteins in DrosophilaSchneider line 2 (S2) cells is also available (InVitrogen, San Diego,Calif.).

[0045] DNA encoding the protease EOS may be subcloned into an expressionvector for expression in a recombinant host cell. Recombinant host cellsmay be prokaryotic or eukaryotic, including but not limited to bacteriasuch as E. coli, fungal cells such as yeast, mammalian cells includingbut not limited to cell lines of human, bovine, porcine, monkey androdent origin, and insect cells including but not limited to DrosophilaS2 (ATCC CRL-1963) and silkworm Sf9 (ATCC CRL-1711), derived cell lines.Cell lines derived from mammalian species which may be suitable andwhich are commercially available, include but are not limited to, CV-1(ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCCCCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL171), L-cells, and HEK-293 (ATCC CRL 1573),

[0046] The expression vector may be introduced into host cells via anyone of a number of techniques including but not limited totransformation, transfection, protoplast fusion, lipofection, andelectroporation. The expression vector-containing cells are clonallypropagated and individually analyzed to determine whether they produceprotease EOS protein. Identification of protease ESO expressing hostcell clones may be done by several means, including but not limited toimmunological reactivity with anti-protease EOS antibodies, and thepresence of host cell-associated EOS proteolytic activity.

[0047] Expression of protease EOS DNA may also be performed using invitro produced synthetic mRNA. Synthetic mRNA or mRNA isolated fromprotease EOS producing cells can be efficiently translated in variouscell-free systems, including but not limited to wheat germ extracts andreticulocyte extracts, as well as efficiently translated in cell basedsystems, including but not limited to microinjection into frog oocytes,with microinjection into frog oocytes being generally preferred.

[0048] To determine the protease EOS DNA sequence(s) that yields optimallevels of EOS proteolytic activity and/or EOS protein, protease EOS DNAmolecules including, but not limited to, the following can beconstructed: the full-length open reading frame of the protease EOS cDNAencoding the 30-kDa protein from approximately base 69 to approximatelybase 920 (these numbers correspond to first nucleotide of firstmethionine and last nucleotide before the first stop codon; FIG. 1) andseveral constructs containing portions of the cDNA encoding the EOSprotease. All constructs can be designed to contain none, all orportions of the 5′ or the 3′ untranslated region of the protease EOScDNA. protease EOS activity and levels of protein expression can bedetermined following the introduction, both singly and in combination,of these constructs into appropriate host cells. Following determinationof the protease EOS DNA cassette yielding optimal expression intransient assays, this protease EOS DNA construct is transferred to avariety of expression vectors, for expression in host cells including,but not limited to, mammalian cells, baculovirus-infected insect cells,E. coli, and the yeast S. cerevisiae.

[0049] Host cell transfectants and microinjected oocytes may be used toassay both the levels of protease EOS proteolytic activity and levels ofEOS protein by the following methods. In the case of recombinant hostcells, this involves the co-transfection of one or possibly two or moreplasmids, containing the protease EOS DNA encoding one or more fragmentsor subunits. In the case of oocytes, this involves the co-injection ofsynthetic RNAs encoding protease EOS. Following an appropriate period oftime to allow for expression, cellular protein is metabolically labeledwith, for example ³⁵S-methionine for 24 hours, after which cell lysatesand cell culture supernatants are harvested and subjected toimmunoprecipitation with polyclonal antibodies directed against theprotease EOS protein.

[0050] Other methods for detecting protease EOS expression involve thedirect measurement of EOS proteolytic activity in whole cellstransfected with protease EOS cDNA or oocytes injected with protease EOSmRNA. Proteolytic activity can be measured by analyzing conditionedmedia or cell lysates by hydrolysis of a chromogenic or fluorogenicsubstrate. In the case of recombinant host cells expressing proteaseEOS, higher levels of substrate hydrolysis would be observed relative tomock transfected cells or cells transfected with expression vectorlacking the protease EOS DNA insert. In the case of oocytes, lysates orconditioned media from those injected with RNA encoding protease EOS,would show higher levels of substrate hydrolysis than those oocytesprogrammed with an irrelevant RNA.

[0051] Other methods for detecting proteolytic activity include, but arenot limited to, measuring the products of proteolytic degradation ofradiolabeled proteins (Coolican et al. (1986). J. Biol. Chem.261:4170-6), fluorometric (Lonergan et al. (1995). J. Food Sci. 60:72-3,78; Twining (1984). Anal. Biochem. 143:30-4) or colorimetric(Buroker-Kilgore and Wang (1993). Anal. Biochem. 208:387-92) analyses ofdegraded protein substrates. Zymography following SDS polyacrylamide gelelectrophoresis (Wadstroem and Smyth (1973). Sci. Tools 20:17-21) , aswell as by fluorescent resonance energy transfer (FRET)-based methods(Ng and Auld (1989). Anal. Biochem. 183:50-6) are also methods used todetect proteolytic activity.

[0052] Levels of protease EOS protein in host cells can be quantitatedby immunoaffinity. protease EOS-specific affinity beads or proteaseEOS-specific antibodies are used to isolate for example ³⁵S-methioninelabeled or unlabelled protease EOS protein. Labeled protease EOS proteinis analyzed by SDS-PAGE. Unlabelled protease EOS protein is detected byWestern blotting, ELISA or RIA assays employing protease EOS specificantibodies.

[0053] Because the genetic code is degenerate, more than one codon maybe used to encode a particular amino acid, and therefore, the amino acidsequence can be encoded by any of a set of similar DNA oligonucleotides.Only one member of the set will be identical to the protease EOSsequence but will be capable of hybridizing to protease EOS DNA even inthe presence of DNA oligonucleotides with mismatches under appropriateconditions. Under alternate conditions, the mismatched DNAoligonucleotides may still hybridize to the protease EOS DNA to permitidentification and isolation of protease EOS encoding DNA.

[0054] DNA encoding protease EOS from a particular organism may be usedto isolate and purify homologues of the protease EOS DNA from otherorganisms. To accomplish this, the first protease EOS DNA may be mixedwith a sample containing DNA encoding homologues of protease EOS underappropriate hybridization conditions. The hybridized DNA complex may beisolated and the DNA encoding the homologous DNA may be purifiedtherefrom.

[0055] It is known that there is a substantial amount of redundancy inthe various codons that code for specific amino acids. Therefore, thisinvention is also directed to those DNA sequences that containalternative codons that code for the eventual translation of theidentical amino acid. For purposes of this specification, a sequencebearing one or more replaced codons will be defined as a degeneratevariation. Also included within the scope of this invention aremutations either in the DNA sequence or the translated protein which donot substantially alter the ultimate physical properties of theexpressed protein. For example, substitution of valine for leucine,arginine for lysine, or asparagine for glutamine may not cause a changein functionality of the polypeptide.

[0056] It is known that DNA sequences coding for a peptide may bealtered so as to code for a peptide having properties that are differentthan those of the naturally occurring peptide. Methods of altering theDNA sequences include, but are not limited to site directed mutagenesis.Examples of altered properties include but are not limited to changes inthe affinity of an enzyme for a substrate or a receptor for a ligand.

[0057] Several recombinant serine protease purification procedures areavailable and suitable for use (Hansson et al. (1994). J. Biol. Chem.269:19420-6; Little et al. (1997). J. Biol. Chem. 272:25135-25142;Takayama et al. (1997). J. Biol. Chem. 272:21582-21588; Yamaoka et al.(1998). J. Biol. Chem. 273:11895-11901). As described above forpurification of protease EOS from natural sources, recombinant proteaseEOS may be purified from cell lysates and extracts, or from conditionedculture medium, by various combinations of, or individual application ofsalt fractionation, ion exchange chromatography, size exclusionchromatography, hydroxylapatite adsorption chromatography andhydrophobic interaction chromatography. Following expression of proteaseEOS in a recombinant host cell, as is the case for many members of theS1 serine protease family, protease EOS protein may be recovered as aninactive zymogen precursor form which may require a limited proteolysisto become the proteolytically active.

[0058] A major drawback in the expression of full-length serine proteasecDNAs for biochemical and enzymological analyses is the overwhelmingpotential for the production of large amounts of the inactive zymogen.These zymogen precursors often have little or no significant proteolyticactivity and thus must be activated by either one of two methodscurrently available. One method relies on the autoactivation (Little etal. (1997). J. Biol. Chem. 272:25135-25142), which may occur inhomogeneous purified protease preparations under the correct set ofcircumstances. Investigators must rigorously evaluate these conditions,which often require high protein concentrations. The second method isthe use of a surrogate activating protease, such as trypsin, to cleavethe serine protease under investigation, and either inactivate (Takayamaet al. (1997). J. Biol. Chem. 272:21582-21588) or physically remove(Hansson et al. (1994). J. Biol. Chem. 269:19420-6) the contaminatingprotease following activation. In both methods however, the exactconditions must be established empirically and activating reactionsmonitored carefully, since inadequate activation or over-digestionleading to degradation and sample loss could always be possibleconsequences of these activating techniques. Investigators studyingparticular members of the S1 serine protease family have exploited theuse of restriction proteinases on the activation of expressed zymogensin bacteria (Wang et al. (1995). Biol. Chem. Hoppe-Seyler 376:681-4) andmammalian cells (Yamashiro et al. (1997). Biochim. Biophys. Acta1350:11-14). In one report, the authors successfully engineered thesecretion of proteolytically processed and activated murine granzyme Bby taking advantage of the endogenous yeast KEX2 signal peptidase in aPichia pastoris expression system (Pham et al. (1998). J. Biol. Chem.273:1629-1633). Another aspect of the present invention provides afusion gene comprising protease EOS that encodes a protease EOS thatfacilitates activation of the protease.

[0059] DNA clones, including protease EOS DNA, are identified whichencode proteins that, when expressed in a recombinant host, produceprotein with the amino acid sequence of protease EOS, which may or maynot possess a proteolytic activity. The expression of protease EOS DNAresults in the reconstitution of the properties observed in oocytesinjected with protease EOS-encoding poly (A)⁺ RNA.

[0060] Recombinant protease EOS can be separated from other cellularproteins by use of an immunoaffinity column made with monoclonal orpolyclonal antibodies specific for full-length nascent protease EOSpolypeptide fragments of protease EOS. Monospecific antibodies toprotease EOS are purified from mammalian antisera containing antibodiesreactive against protease EOS or are prepared as monoclonal antibodiesreactive with protease EOS using the technique of (Kohler and Milstein(1976). Eur J Immunol 6:511-9). Monospecific antibody as used herein isdefined as a single antibody species or multiple antibody species withhomogenous binding characteristics for protease EOS. Homogenous bindingas used herein refers to the ability of the antibody species to bind toa specific antigen or epitope, such as those associated with theprotease EOS, as described above. Protease EOS specific antibodies areraised by immunizing animals such as mice, rats, guinea pigs, rabbits,goats, horses and the like, with rabbits being preferred, with anappropriate concentration of protease EOS either with or without animmune adjuvant.

[0061] Preimmune serum is collected prior to the first immunization.Each animal receives between about 0.1 mg and about 1000 mg of proteaseEOS protein or peptide(s), derived from the deduced protease EOS DNAsequence or perhaps by the chemical degradation or enzymatic digestionof the protease EOS protein itself, associated with an acceptable immuneadjuvant. Such acceptable adjuvants include, but are not limited to,Freund's complete, Freund's incomplete, alum-precipitate, water in oilemulsion containing Corynebacterium parvum and tRNA, or Titermax (CytRx,Norcross, Ga.). The initial immunization consists of protease EOSantigen in, preferably, Freund's complete adjuvant at multiple siteseither subcutaneously (SC), intraperitoneally (IP) or both. Each animalis bled at regular intervals, preferably weekly, to determine antibodytiter. The animals may or may not receive booster injections followingthe initial immunization. Those animals receiving booster injections aregenerally given an equal amount of the antigen in Freund's incompleteadjuvant by the same route. Booster injections are given at aboutthree-week intervals until maximal titers are obtained. At about 7 daysafter each booster immunization or about weekly after a singleimmunization, the animals are bled, the serum collected, and aliquotsare stored at about −20° C.

[0062] Monoclonal antibodies (MoAb) reactive with protease EOS areprepared by immunizing inbred mice, preferably Balb/c, with protease EOSprotein or peptide(s), derived from the deduced protease EOS DNAsequence or perhaps by the chemical degradation or enzymatic digestionof the protease EOS protein itself. The mice are immunized by the IP orSC route with about 0.1 mg to about 10 mg, preferably about 1 mg, ofprotease EOS antigen in about 0.5 ml buffer or saline incorporated in anequal volume of an acceptable adjuvant, as discussed above. Freund'scomplete adjuvant is preferred. The mice receive an initial immunizationon day 0 and are rested for about 3 to about 30 weeks. Immunized miceare given one or more booster immunizations of about 0.1 to about 10 mgof protease EOS antigen in a buffer solution such as phosphate bufferedsaline by the intravenous (IV) route. Lymphocytes, from antibodypositive mice, preferably splenic lymphocytes, are obtained by removingspleens from immunized mice by standard procedures known in the art.Hybridoma cells are produced by mixing the splenic lymphocytes with anappropriate fusion partner, preferably myeloma cells, under conditionsthat will allow the formation of stable hybridomas. Fusion partners mayinclude, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11;S-194 and Sp 2/0, with Sp 2/0 being generally preferred. The antibodyproducing cells and myeloma cells are fused in polyethylene glycol,about 1000 mol. wt., at concentrations from about 30% to about 50%.Fused hybridoma cells are selected by growth in hypoxanthine, thymidineand aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) byprocedures known in the art. Supernatant fluids are collected fromgrowth positive wells on about days 14, 18, and 21 and are screened forantibody production by an immunoassay such as solid phaseimmunoradioassay (SPIRA) using protease EOS or antigenic peptide(s) asthe antigen. The culture fluids are also tested in the Ouchterlonyprecipitation assay to determine the isotype of the MoAb. Hybridomacells from antibody positive wells are cloned by a technique such as thesoft agar technique of MacPherson, Soft Agar Techniques, in TissueCulture Methods and Applications, Kruse and Paterson, Eds., AcademicPress, 1973.

[0063] Monoclonal antibodies are produced in vivo by injection ofpristane primed Balb/c mice, approximately 0.5 ml per mouse, with about2×10⁶ to about 6×10⁶ hybridoma cells about 4 days after priming. Ascitesfluid is collected at approximately 8-12 days after cell transfer andthe monoclonal antibodies are purified by techniques known in the art.

[0064] In vitro production of anti-protease EOS MoAb is carried out bygrowing the hybridoma in DMEM containing about 2% fetal calf serum toobtain sufficient quantities of the specific MoAb. The monoclonalantibodies are purified by techniques known in the art.

[0065] Antibody titers of ascites or hybridoma culture fluids aredetermined by various serological or immunological assays which include,but are not limited to, precipitation, passive agglutination,enzyme-linked immunosorbent antibody (ELISA) technique andradioimmunoassay (RIA) techniques. Similar assays are used to detect thepresence of protease EOS in body fluids or tissue and cell extracts.

[0066] It is readily apparent to those skilled in the art that the abovedescribed methods for producing monospecific antibodies may be utilizedto produce antibodies specific for protease EOS polypeptide fragments,or full-length nascent protease EOS polypeptide. Specifically, it isreadily apparent to those skilled in the art that monospecificantibodies may be generated which are specific for only one or moreprotease EOS epitopes.

[0067] Protease EOS antibody affinity columns are made by adding theantibodies to Affigel-10 (Bio-Rad), a gel support which is activatedwith N-hydroxysuccinimide esters such that the antibodies form covalentlinkages with the agarose gel bead support. The antibodies are thencoupled to the gel via amide bonds with the spacer arm. The remainingactivated esters are then quenched with 1M ethanolamine HCl (pH 8). Thecolumn is washed with water followed by 0.23 M glycine HCl (pH 2.6) toremove any non-conjugated antibody or extraneous protein. The column isthen equilibrated in phosphate buffered saline (pH 7.3) and the cellculture supernatants or cell extracts containing protease EOS are slowlypassed through the column. The column is then washed with phosphatebuffered saline until the optical density (A₂₈₀) falls to background,then the protein is eluted with 0.23 M glycine-HCl (pH 2.6). Thepurified protease EOS protein is then dialyzed against phosphatebuffered saline.

[0068] Protease EOS mRNA is expressed in retina, ovary, and stomach,where the encoded protease EOS protein may perform important functionsduring normal physiology, and possibly pathological states. Thus,modulators of protease EOS function could be used to treat disorderseffecting these tissues. We have also found that the protease EOS mRNAis expressed human platelets, and spleen which is a major site of bloodcell metabolism, as well as in leukocytes and more specifically ineosinophils. Eosinophilia, is a condition characterized by elevatedcirculating eosinophils, and is associated with numerous allergic statesincluding bronchial asthma (Gleich (1996). Allergol. Int. 45:35-44).Thus, protease EOS expression in platelets and certain cells of theimmune system suggests that it may play roles in hemostasis and a subsetof immune processes. Modulators of protease EOS function could thereforepotentially be used to treat disorders in hemostasis and/or to moderateparticular immune responses.

[0069] The present invention is also directed to methods for screeningfor compounds that modulate the expression of DNA or RNA encodingprotease T as well as the function of protease T protein in vivo.Compounds that modulate these activities may be DNA, RNA, peptides,proteins, or non-proteinaceous organic molecules. Compounds may modulateby increasing or attenuating the expression of DNA or RNA encodingprotease T, or the function of protease T protein. Compounds thatmodulate the expression of DNA or RNA encoding protease T or thefunction of protease T protein may be detected by a variety of assays.The assay may be a simple “yes/no” assay to determine whether there is achange in expression or function. The assay may be made quantitative bycomparing the expression or function of a test sample with the levels ofexpression or function in a standard sample. Modulators identified inthis process are potentially useful as therapeutic agents. Methods fordetecting compounds that modulate protease T proteolytic activitycomprise combining compound, protease T and a suitable labeled substrateand monitoring an effect of the compound on the protease by changes inthe around of substrate as a function of time. Labeled substratesinclude, but are not limited to, substrate that are radiolabeled(Coolican et al. (1986). J. Biol. Chem. 261:4170-6), fluorometric(Lonergan et al. (1995). J. Food Sci. 60:72-3, 78; Twining (1984). Anal.Biochem. 143:30-4) or calorimetric (Buroker-Kilgore and Wang (1993).Anal. Biochem. 208:387-92). Zymography following SDS polyacrylamide gelelectrophoresis (Wadstroem and Smyth (1973). Sci. Tools 20:17-21), aswell as by fluorescent resonance energy transfer (FRET)-based methods(Ng and Auld (1989). Anal. Biochem. 183:50-6) are also methods used todetect compounds that modulate protease T proteolytic activity.Compounds that are agonists will increase the rate of substratedegradation and will result in less remaining substrate as a function oftime. Compounds that are antagonists will decrease the rate of substratedegradation and will result in greater remaining substrate as a functionof time.

[0070] Kits containing protease EOS DNA or RNA, antibodies to proteaseEOS, or protease EOS protein may be prepared. Such kits are used todetect DNA that hybridizes to protease EOS DNA or to detect the presenceof protease EOS protein or peptide fragments in a sample. Suchcharacterization is useful for a variety of purposes including but notlimited to forensic analyses, diagnostic applications, andepidemiological studies.

[0071] The present invention is also directed to methods for screeningfor compounds that modulate the expression of DNA or RNA encodingprotease EOS as well as the function of protease EOS protein in vivo.Compounds that modulate these activities may be DNA, RNA, peptides,proteins, or non-proteinaceous organic molecules. Compounds may modulateby increasing or attenuating the expression of DNA or RNA encodingprotease EOS, or the function of protease EOS protein. Compounds thatmodulate the expression of DNA or RNA encoding protease EOS or thefunction of protease EOS protein may be detected by a variety of assays.The assay may be a simple “yes/no” assay to determine whether there is achange in expression or function. The assay may be made quantitative bycomparing the expression or function of a test sample with the levels ofexpression or function in a standard sample. Modulators identified inthis process are potentially useful as therapeutic agents. Methods fordetecting compounds that modulate protease EOS proteolytic activitycomprise combining compound, protease EOS and a suitable labeledsubstrate and monitoring an effect of the compound on the protease bychanges in the amount of substrate as a function of time. Labeledsubstrates include, but are not limited to, substrates that areradiolabeled (Coolican et al. (1986). J. Biol. Chem. 261:4170-6),fluorometric (Lonergan et al. (1995). J. Food Sci. 60:72-3, 78; Twining(1984). Anal. Biochem. 143:30-4) or calorimetric (Buroker-Kilgore andWang (1993). Anal. Biochem. 208:387-92). Zymography following SDSpolyacrylamide gel electrophoresis (Wadstroem and Smyth (1973). Sci.Tools 20:17-21), as well as by fluorescent resonance energy transfer(FRET)-based methods (Ng and Auld (1989). Anal. Biochem. 183:50-6) arealso methods used to detect compounds that modulate protease EOSproteolytic activity. Compounds that are agonists will increase the rateof substrate degradation and will result in less remaining substrate asa function of time. Compounds that are antagonists will decrease therate of substrate degradation and will result in greater remainingsubstrate as a function of time.

[0072] Nucleotide sequences that are complementary to the protease EOSencoding DNA sequence can be synthesized for antisense therapy. Theseantisense molecules may be DNA, stable derivatives of DNA such asphosphorothioates or methylphosphonates, RNA, stable derivatives of RNAsuch as 2′-O-alkylRNA, or other protease EOS antisense oligonucleotidemimetics. protease EOS antisense molecules may be introduced into cellsby microinjection, liposome encapsulation or by expression from vectorsharboring the antisense sequence. protease EOS antisense therapy may beparticularly useful for the treatment of diseases where it is beneficialto reduce protease EOS expression or activity.

[0073] Protease EOS gene therapy may be used to introduce protease EOSinto the cells of target organisms. The protease EOS gene can be ligatedinto viral vectors that mediate transfer of the protease EOS DNA byinfection of recipient host cells. Suitable viral vectors includeretrovirus, adenovirus, adeno-associated virus, herpes virus, vacciniavirus, poliovirus and the like. Alternatively, protease EOS DNA can betransferred into cells for gene therapy by non-viral techniquesincluding receptor-mediated targeted DNA transfer using ligand-DNAconjugates or adenovirus-ligand-DNA conjugates, lipofection membranefusion or direct microinjection. These procedures and variations thereofare suitable for ex vivo as well as in vivo protease EOS gene therapy.Protease EOS gene therapy may be particularly useful for the treatmentof diseases where it is beneficial to elevate protease EOS expression oractivity.

[0074] Pharmaceutically useful compositions comprising protease EOS DNA,protease EOS RNA, or protease EOS protein, or modulators of protease EOSactivity, may be formulated according to known methods such as by theadmixture of a pharmaceutically acceptable carrier. Examples of suchcarriers and methods of formulation may be found in Remington'sPharmaceutical Sciences. To form a pharmaceutically acceptablecomposition suitable for effective administration, such compositionswill contain an effective amount of the protein, DNA, RNA, or modulator.

[0075] Therapeutic or diagnostic compositions of the invention areadministered to an individual in amounts sufficient to treat or diagnosedisorders in which modulation of protease EOS-related activity isindicated. The effective amount may vary according to a variety offactors such as the individual's condition, weight, sex and age. Otherfactors include the mode of administration. The pharmaceuticalcompositions may be provided to the individual by a variety of routessuch as subcutaneous, topical, oral and intramuscular.

[0076] The term “chemical derivative” describes a molecule that containsadditional chemical moieties that are not normally a part of the basemolecule. Such moieties may improve the solubility, half-life,absorption, etc. of the base molecule. Alternatively the moieties mayattenuate undesirable side effects of the base molecule or decrease thetoxicity of the base molecule. Examples of such moieties are describedin a variety of texts, such as Remington's Pharmaceutical Sciences.

[0077] Compounds identified according to the methods disclosed hereinmay be used alone at appropriate dosages defined by routine testing inorder to obtain optimal inhibition of the protease EOS activity whileminimizing any potential toxicity. In addition, co-administration orsequential administration of other agents may be desirable.

[0078] The protease EOS may be formulated as an active ingredient innon-pharmaceutical commercial products including laundry detergents,skin care lotions or creams. In these formulations the protease EOS isutilized to degrade proteins to increase the efficacy of the product.For example, in laundry detergent formulations inclusion of the proteaseEOS would act as a “stain remover” by degrading proteacious contaminantsfrom fabric such that the organic compound would become more soluble indetergent and water. Protease EOS can be included in skin care productsto aid in desquamation, the process of elimination of the superficiallayers of the stratum corneum. An additional benefit of utilizing theprotease EOS in non-pharmaceutical commercial formulations is that it isnot likely to induce allergic response in sensitive individuals sincethe protease EOS is of human origin.

[0079] The present invention also has the objective of providingsuitable topical, oral, systemic and parenteral pharmaceuticalformulations for use in the novel methods of treatment of the presentinvention. The compositions containing compounds or modulatorsidentified according to this invention as the active ingredient for usein the modulation of protease EOS activity can be administered in a widevariety of therapeutic dosage forms in conventional vehicles foradministration. For example, the compounds or modulators can beadministered in such oral dosage forms as tablets, capsules (eachincluding timed release and sustained release formulations), pills,powders, granules, elixirs, tinctures, solutions, suspensions, syrupsand emulsions, or by injection. Likewise, they may also be administeredin intravenous (both bolus and infusion), intraperitoneal, subcutaneous,topical with or without occlusion, or intramuscular form, all usingforms well known to those of ordinary skill in the pharmaceutical arts.An effective but non-toxic amount of the compound desired can beemployed as a protease EOS modulating agent.

[0080] The daily dosage of the products may be varied over a wide rangefrom 0.01 to 1,000 mg per patient, per day. For oral administration, thecompositions are preferably provided in the form of scored or unscoredtablets containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0,25.0, and 50.0 milligrams of the active ingredient for the symptomaticadjustment of the dosage to the patient to be treated. An effectiveamount of the drug is ordinarily supplied at a dosage level of fromabout 0.0001 mg/kg to about 100 mg/kg of body weight per day. The rangeis more particularly from about 0.001 mg/kg to 10 mg/kg of body weightper day. The dosages of the protease EOS modulators are adjusted whencombined to achieve desired effects. On the other hand, dosages of thesevarious agents may be independently optimized and combined to achieve asynergistic result wherein the pathology is reduced more than it wouldbe if either agent were used alone.

[0081] Advantageously, compounds or modulators of the present inventionmay be administered in a single daily dose, or the total daily dosagemay be administered in divided doses of two, three or four times daily.Furthermore, compounds or modulators for the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen.

[0082] For combination treatment with more than one active agent, wherethe active agents are in separate dosage formulations, the active agentscan be administered concurrently, or they each can be administered atseparately staggered times.

[0083] The dosage regimen utilizing the compounds or modulators of thepresent invention is selected in accordance with a variety of factorsincluding type, species, age, weight, sex and medical condition of thepatient; the severity of the condition to be treated; the route ofadministration; the renal and hepatic function of the patient; and theparticular compound thereof employed. A physician or veterinarian ofordinary skill can readily determine and prescribe the effective amountof the drug required to prevent, counter or arrest the progress of thecondition. Optimal precision in achieving concentrations of drug withinthe range that yields efficacy without toxicity requires a regimen basedon the kinetics of the drug's availability to target sites. Thisinvolves a consideration of the distribution, equilibrium, andelimination of a drug.

[0084] In the methods of the present invention, the compounds ormodulators herein described in detail can form the active ingredient,and are typically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

[0085] For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, disintegrating agents and coloring agentscan also be incorporated into the mixture. Suitable binders include,without limitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, sodium chloride andthe like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum and the like.

[0086] For liquid forms the active drug component can be combined insuitably flavored suspending or dispersing agents such as the syntheticand natural gums, for example, tragacanth, acacia, methyl-cellulose andthe like. Other dispersing agents that may be employed include glycerinand the like. For parenteral administration, sterile suspensions andsolutions are desired. Isotonic preparations, which generally containsuitable preservatives, are employed when intravenous administration isdesired.

[0087] Topical preparations containing the active drug component can beadmixed with a variety of carrier materials well known in the art, suchas, eg., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and Eoils, mineral oil, PPG2 myristyl propionate, and the like, to form, eg.,alcoholic solutions, topical cleansers, cleansing creams, skin gels,skin lotions, and shampoos in cream or gel formulations.

[0088] The compounds or modulators of the present invention can also beadministered in the form of liposome delivery systems, such as smallunilamellar vesicles, large unilamellar vesicles and multilamellarvesicles. Liposomes can be formed from a variety of phospholipids, suchas cholesterol, stearylamine or phosphatidylcholines.

[0089] Compounds of the present invention may also be delivered by theuse of monoclonal antibodies as individual carriers to which thecompound molecules are coupled. The compounds or modulators of thepresent invention may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxy-ethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds ormodulators of the present invention may be coupled to a class ofbiodegradable polymers useful in achieving controlled release of a drug,for example, polylactic acid, polyepsilon caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydro-pyrans,polycyanoacrylates and cross-linked or amphipathic block copolymers ofhydrogels.

[0090] For oral administration, the compounds or modulators may beadministered in capsule, tablet, or bolus form or alternatively they canbe mixed in the animals feed. The capsules, tablets, and boluses arecomprised of the active ingredient in combination with an appropriatecarrier vehicle such as starch, talc, magnesium stearate, or di-calciumphosphate. These unit dosage forms are prepared by intimately mixing theactive ingredient with suitable finely-powdered inert ingredientsincluding diluents, fillers, disintegrating agents, and/or binders suchthat a uniform mixture is obtained. An inert ingredient is one that willnot react with the compounds or modulators and which is non-toxic to theanimal being treated. Suitable inert ingredients include starch,lactose, talc, magnesium stearate, vegetable gums and oils, and thelike. These formulations may contain a widely variable amount of theactive and inactive ingredients depending on numerous factors such asthe size and type of the animal species to be treated and the type andseverity of the infection. The active ingredient may also beadministered as an additive to the feed by simply mixing the compoundwith the feedstuff or by applying the compound to the surface of thefeed. Alternatively the active ingredient may be mixed with an inertcarrier and the resulting composition may then either be mixed with thefeed or fed directly to the animal. Suitable inert carriers include cornmeal, citrus meal, fermentation residues, soya grits, dried grains andthe like. The active ingredients are intimately mixed with these inertcarriers by grinding, stirring, milling, or tumbling such that the finalcomposition contains from 0.001 to 5% by weight of the activeingredient.

[0091] The compounds or modulators may alternatively be administeredparenterally via injection of a formulation consisting of the activeingredient dissolved in an inert liquid carrier. Injection may be eitherintramuscular, intraruminal, intratracheal, or subcutaneous. Theinjectable formulation consists of the active ingredient mixed with anappropriate inert liquid carrier. Acceptable liquid carriers include thevegetable oils such as peanut oil, cottonseed oil, sesame oil and thelike as well as organic solvents such as solketal, glycerol formal andthe like. As an alternative, aqueous parenteral formulations may also beused. The vegetable oils are the preferred liquid carriers. Theformulations are prepared by dissolving or suspending the activeingredient in the liquid carrier such that the final formulationcontains from 0.005 to 10% by weight of the active ingredient.

[0092] Topical application of the compounds or modulators is possiblethrough the use of a liquid drench or a shampoo containing the instantcompounds or modulators as an aqueous solution or suspension. Theseformulations generally contain a suspending agent such as bentonite andnormally will also contain an antifoaming agent. Formulations containingfrom 0.005 to 10% by weight of the active ingredient are acceptable.Preferred formulations are those containing from 0.01 to 5% by weight ofthe instant compounds or modulators.

[0093] Proteases are used in non-natural environments for variouscommercial purposes including laundry detergents, food processing,fabric processing, and skin care products. In laundry detergents, theprotease is employed to break down organic, poorly soluble compounds tomore soluble forms that can be more easily dissolved in detergent andwater. In this capacity the protease acts as a “stain remover.” Examplesof food processing include tenderizing meats and producing cheese.Proteases are used in fabric processing, for example, to treat wool inorder prevent fabric shrinkage. Proteases may be included in skin careproducts to remove scales on the skin surface that build up due to animbalance in the rate of desquamation. Common proteases used in some ofthese applications are derived from prokaryotic or eukaryotic cells thatare easily grown for industrial manufacture of their enzymes, forexample a common species used is Bacillus as described in U.S. Pat. No.5,217,878. Alternatively, U.S. Pat. No. 5,278,062 describes serineproteases isolated from a fungus, Tritirachium album, for use in laundrydetergent compositions. Unfortunately use of some proteases is limitedby their potential to cause allergic reactions in sensitive individualsor by reduced efficiency when used in a non-natural environment. It isanticipated that protease proteins derived from non-human sources wouldbe more likely to induce an immune response in a sensitive individual.Because of these limitations, there is a need for alternative proteasesthat are less immunogenic to sensitive individuals and/or providesefficient proteolytic activity in a non-natural environment. The adventof recombinant technology allows expression of any species' proteins ina host suitable for industrial manufacture.

[0094] Another aspect of the present invention relates to compositionscomprising the protease EOS and an acceptable carrier. The compositionmay be any variety of compositions that requires a protease component.Particularly preferred are compositions that may come in contact withhumans, for example, through use or manufacture. The use of the proteaseEOS of the present invention is believed to reduce or eliminate theimmunogenic response users and/or handlers might otherwise experiencewith a similar composition containing a known protease, particularly aprotease of non-human origin. Preferred compositions are skin carecompositions and laundry detergent compositions.

[0095] Herein, “acceptable carriers” includes, but is not limited to,cosmetically-acceptable carriers, pharmaceutically-acceptable carriers,and carriers acceptable for use in cleaning compositions.

[0096] Skin Care Compositions

[0097] Skin care compositions of the present invention preferablycomprise, in addition to the protease EOS, a cosmetically- orpharmaceutically-acceptable carrier.

[0098] Herein, “cosmetically-acceptable carrier” means one or morecompatible solid or liquid filler diluents or encapsulating substanceswhich are suitable for use in contact with the skin of humans and loweranimals without undue toxicity, incompatibility, instability,irritation, allergic response, and the like, commensurate with areasonable benefit/risk ratio.

[0099] Herein, “pharmaceutically-acceptable” means one or morecompatible drugs, medicaments or inert ingredients which are suitablefor use in contact with the tissues of humans and lower animals withoutundue toxicity, incompatibility, instability, irritation, allergicresponse, and the like, commensurate with a reasonable. benefit/riskratio. Pharmaceutically-acceptable carriers must, of course, be ofsufficiently high purity and sufficiently low toxicity to render themsuitable for administration to the mammal being treated.

[0100] Herein, “compatible” means that the components of the cosmetic orpharmaceutical compositions are capable of being commingled with theprotease EOS, and with each other, in a manner such that there is nointeraction which would substantially reduce the cosmetic orpharmaceutical efficacy of the composition under ordinary usesituations.

[0101] Preferably the skin care compositions of the present inventionare topical compositions, i.e., they are applied topically by the directlaying on or spreading of the composition on skin. Preferably suchtopical compositions comprise a cosmetically- orpharmaceutically-acceptable topical carrier.

[0102] The topical composition may be made into a wide variety ofproduct types. These include, but are not limited to, lotions, creams,beach oils, gels, sticks, sprays, ointments, pastes, mousses, andcosmetics; hair care compositions such as shampoos and conditioners(for, e.g., treating/preventing dandruff); and personal cleansingcompositions. These product types may comprise several carrier systemsincluding, but not limited to, solutions, emulsions, gels and solids.

[0103] Preferably the carrier is a cosmetically- orpharmaceutically-acceptable aqueous or organic solvent. Water is apreferred solvent. Examples of suitable organic solvents include:propylene glycol, polyethylene glycol (200-600), polypropylene glycol(425-2025), propylene glycol-14 butyl ether, glycerol, 1,2,4butanetriol,sorbitol esters, 1,2,6-hexanetriol, ethanol, isopropanol, butanediol,and mixtures thereof. Such solutions useful in the present inventionpreferably contain from about 0.001% to about 25% of the protease EOS,more preferably from about 0.1% to about 10% more preferably from about0.5% to about 5%; and preferably from about 50% to about 99.99% of anacceptable aqueous or organic solvent, more preferably from about 90% toabout 99%.

[0104] Skin care compositions of the present invention may furtherinclude a wide variety of additional oil-soluble materials and/orwater-soluble materials conventionally used in topical compositions, attheir art-established levels. Such additional components include, butare not limited to: thickeners, pigments, fragrances, humectants,proteins and polypeptides, preservatives, pacifiers, penetrationenhancing agents, collagen, hylauronic acid, elastin, hydrolysates,primrose oil, jojoba oil, epidermal growth factor, soybean saponins,mucopolysaccharides, Vitamin A and derivatives thereof, Vitamin B2,biotin, pantothenic acid, Vitamin D, and mixtures thereof.

[0105] Cleaning Compositions

[0106] Cleaning compositions of the present invention preferablycomprise, in addition to the protease EOS, a surfactant. The cleaningcomposition may be in a wide variety of forms, including, but notlimited to, hard surface cleaning compositions, dishcare cleaningcompositions, and laundry detergent compositions.

[0107] Preferred cleaning compositions are laundry detergentcompositions. Such laundry detergent compositions include, but notlimited to, granular, liquid and bar compositions. Preferably, thelaundry detergent composition further comprises a builder.

[0108] The laundry detergent composition of the present inventioncontains the protease EOS at a level sufficient to provide a“cleaning-effective amount”. The term “cleaning effective amount” refersto any amount capable of producing a cleaning, stain removal, soilremoval, whitening, deodorizing, or freshness improving effect onsubstrates such as fabrics, dishware and the like. In practical termsfor current commercial preparations, typical amounts are up to about 5mg by weight, more typically 0.01 mg to 3 mg, of active enzyme per gramof the detergent composition. Stated another way, the laundry detergentcompositions herein will typically comprise from 0.001% to 5%,preferably 0.01%-3%, more preferably 0.01% to 1% by weight of rawprotease EOS preparation. Herein, “raw protease EOS preparation” refersto preparations or compositions in which the protease EOS is containedin prior to its addition to the laundry detergent composition.Preferably, the protease EOS is present in such raw protease EOSpreparations at levels sufficient to provide from 0.005 to 0.1 Ansonunits (AU) of activity per gram of raw protease EOS preparation. Forcertain detergents, such as in automatic dishwashing, it maybe desirableto increase the active protease EOS content of the raw protease EOSpreparation in order to minimize the total amount of non-catalyticallyactive materials and thereby improve spotting/filming or otherend-results. Higher active levels may also be desirable in highlyconcentrated detergent formulations.

[0109] Preferably, the laundry detergent compositions of the presentinvention, including but not limited to liquid compositions, maycomprise from about 0.001% to about 10%, preferably from about 0.005% toabout 8%, most preferably from about 0.01% to about 6%, by weight of anenzyme stabilizing system. The enzyme stabilizing system can be anystabilizing system that is compatible with the protease EOS, or anyother additional detersive enzymes that may be included in thecomposition. Such a system may be inherently provided by otherformulation actives, or be added separately, e.g., by the formulator orby a manufacturer of detergent-ready enzymes. Such stabilizing systemscan, for example, comprise calcium ion, boric acid, propylene glycol,short chain carboxylic acids, boronic acids, and mixtures thereof, andare designed to address different stabilization problems depending onthe type and physical form of the detergent composition.

[0110] The detergent composition also comprises a detersive surfactant.Preferably the detergent composition comprises at least about 0.01% of adetersive surfactant; more preferably at least about 0.1%; morepreferably at least about 1%; more preferably still, from about 1% toabout 55%.

[0111] Preferred detersive surfactants are cationic, anionic, nonionic,ampholytic, zwitterionic, and mixtures thereof, further described hereinbelow. Nonlimiting examples of detersive surfactants useful in thedetergent composition include, the conventional C11-C18 alkyl benzenesulfonates (“LAS”) and primary, branched-chain and random C10-C20 alkylsulfates (“AS”), the C10-C18 secondary (2,3) alkyl sulfates of theformula CH₃(CH₂)x(CHOSO₃-M+)CH₃ and CH₃ (CH₂)_(y) (CHOSO₃-M+)CH₂CH₃where x and (y+1) are integers of at least about 7, preferably at leastabout 9, and M is a water-solubilizing cation, especially sodium,unsaturated sulfates such as oleyl sulfate, the C10-C18 alkyl alkoxysulfates (“AExS”; especially EO 1-7 ethoxy sulfates), C10-C18 alkylalkoxy carboxylates (especially the EO 1-5 ethoxycarboxylates), theC10-18 glycerol ethers, the C10-C18 alkyl polyglycosides and theircorresponding sulfated polyglycosides, and C12-C18 alpha-sulfonatedfatty acid esters. If desired, the conventional nonionic and amphotericsurfactants such as the C12-C18 alkyl ethoxylates (“AE”) including theso-called narrow peaked alkyl Ethoxylates and C6-C12 alkyl phenolalkoxylates (especially ethoxylates and mixed ethoxy/propoxy), C12-C18betaines and solfobetaines (“sultaines”), C10-C18 amine oxides, and thelike, can also be included in the overall compositions. The C10-C18N-alkyl polyhydroxy fatty acid amides can also be used. Typical examplesinclude the C12-C18 N-methylglucamides. See WO 9,206,154. Othersugar-derived surfactants include the N-alkoxy polyhydroxy fatty acidamides, such as C10-C18 N-(3-methoxypropyl) glucamide. The N-propylthrough N-hexyl C12-C18 glucamides can be used for low sudsing. C10-C20conventional soaps may also be used. If high sudsing is desired, thebranched-chain C10-C16 soaps may be used. Mixtures of anionic andnonionic surfactants are especially useful. Other conventional usefulsurfactants are listed in standard texts. Detergent builders are alsoincluded in the laundry detergent composition to assist in controllingmineral hardness. Inorganic as well as organic builders can be used.Builders are typically used in fabric laundering compositions to assistin the removal of particulate soils.

[0112] The level of builder can vary widely depending upon the end useof the composition and its desired physical form. When present, thecompositions will typically comprise at least about 1% builder. Liquidformulations typically comprise from about 5% to about 50%, moretypically about 5% to about 30%, by weight, of detergent builder.Granular formulations typically comprise from about 10% to about 80%,more typically from about 15% to about 50% by weight, of the detergentbuilder. Lower or higher levels of builder, however, are not meant to beexcluded.

[0113] Inorganic or P-containing detergent builders include, but are notlimited to, the alkali metal, ammonium and alkanolammonium salts ofpolyphosphates (exemplified by the tripolyphosphates, pyrophosphates,and glassy polymeric meta-phosphates), phosphonates, phytic acid,silicates, carbonates (including bicarbonates and sesquicarbonates),sulphates, and aluminosilicates. However, non-phosphate builders arerequired in some locales. Importantly, the compositions herein functionsurprisingly well even in the presence of the so-called “weak” builders(as compared with phosphates) such as citrate, or in the so-called“underbuilt” situation that may occur with zeolite or layered silicatebuilders.

[0114] Examples of silicate builders are the alkali metal silicates,particularly those having a SiO2:Na2O ration in the range 1.6:1 to 3.2:1and layered silicates, such as the layered sodium silicates described inU.S. Pat. No. 4,664,839, issued May 12, 1987 to H. P. Rieck. NaSKS-6 isthe trademark for a crystalline layered silicate marketed by Hoechst(commonly abbreviated herein as “SKS-6”). Unlike zeolite builders, theNa SKS-6 silicate builder does not contain aluminum. NaSKS-6 has thedelta-Na2SiO5 morphology form of layered silicate. It can be prepared bymethods such as those described in German DE-A-3,417,649 andDE-A-3,742,043. SKS-6 is a highly preferred layered silicate for useherein, but other such layered silicates, such as those having thegeneral formula NaMSixO2x+1 yH20 wherein M is sodium or hydrogen, x is anumber from 1.9 to 4, preferably 2, and y is a number from 0 to 20,preferably 0 can be used herein. Various other layered silicates fromHoechst include NaSKS-5, NaSKS-7 and NaSKS-11, as the alpha, beta andgamma forms. As noted above, the delta-Na2SiO5 (NaSKS-6 form) is mostpreferred for use herein. Other silicates may also be useful such as forexample magnesium silicate, which can serve as a crispening agent ingranular formulations, as a stabilizing agent for oxygen bleaches, andas a component of suds control systems.

[0115] Examples of carbonate builders are the alkaline earth and alkalimetal carbonates as disclosed in German Patent Application No. 2,321,001published on Nov. 15, 1973.

[0116] Aluminosilicate builders are useful in the present invention.Aluminosilicate builders are of great importance in most currentlymarketed heavy duty granular detergent compositions, and can also be asignificant builder ingredient in liquid detergent formulations.Aluminosilicate builders include those having the empirical formula:

M_(z) (zAl0₂)_(y)-xH₂0

[0117] wherein z and y are integers of at least 6, the molar ratio of zto y is in the range from 1.0 to about 0.5, and x is an integer fromabout 15 to about 264.

[0118] Useful aluminosilicate ion exchange materials are commerciallyavailable. These aluminosilicates can be crystalline or amorphous instructure and can be naturally-occurring aluminosilicates orsynthetically derived. A method for producing aluminosilicate ionexchange materials is disclosed in U.S. Pat. No. 3,985,669, Krummel, etal, issued Oct. 12, 1976. Preferred synthetic crystallinealuminosilicate ion exchange materials useful herein are available underthe designations Zeolite A, Zeolite P (b), Zeolite MAP and Zeolite X. Inan especially preferred embodiment, the crystalline aluminosilicate ionexchange material has the formula:

Na₁₂[Al0₂)₁₂(SiO₂)₁₂].xH₂0

[0119] wherein x is from about 20 to about 30, especially about 27. Thismaterial is known as Zeolite A. Dehydrated zeolites (x=0−10) may also beused herein. Preferably, the aluminosilicate has a particle size ofabout 0.1-10 microns in diameter.

[0120] Organic detergent builders suitable for the purposes of thepresent invention include, but are not restricted to, a wide variety ofpolycarboxylate compounds. As used herein, “polycarboxylate” refers tocompounds having a plurality of carboxylate groups, preferably at least3 carboxylates. Polycarboxylate builder can generally be added to thecomposition in acid form, but can also be added in the form of aneutralized salt. When utilized in salt form, alkali metals, such assodium, potassium, and lithium, or alkanolammonium salts are preferred.

[0121] Included among the polycarboxylate builders are a variety ofcategories of useful materials. One important category ofpoiycarboxylate builders encompasses the ether polycarboxylates,including oxydisuccinate, as disclosed in Berg, U.S. Pat. No. 3,128,287,issued Apr. 7, 1964, and Lamberti et al., U.S. Pat. No. 3,635,830,issued Jan. 18, 1972. See also “TMSFTDS” builders of U.S. Pat. No.4,663,071, issued to Bush et al., on May 5, 1987. Suitable etherpolycarboxylates also include cyclic compounds, particularly alicycliccompounds, such as those described in U.S. Pat. No. 3,923,679 to Rapko,issued Dec. 2, 1975; U.S. Pat. No. 3,835,163 to Rapko, issued Sep. 10,1974; U.S. Pat. No. 4,158,635 to Crutchfield et al., issued Jun. 19,1979; U.S. Pat. No. 4,120,874 to Crutchfield et al., issued Oct. 17,1978; and U.S. Pat. No. 4,102,903 to Crutchfield et al., issued Jul. 25,1978.

[0122] Other useful detergency builders include the etherhydroxypolycarboxylates, copolymers of maleic anhydride with ethylene orvinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-t6sulphonic acid, andcarboxymethyloxysuccinic acid, the various alkali metal, ammonium andsubstituted ammonium salts of polyacetic acids such as ethylenediaminetetraacetic acid and nitrilotriacetic acid, as well as polycarboxylatessuch as Mellitic acid, succinic acid, oxydisuccinic acid, polymaleicacid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid,and soluble salts thereof, citrate builders, e.g., citric acid andsoluble salts thereof (particularly sodium salt), are polycarboxylatebuilders of particular importance for heavy-duty liquid detergentformulations due to their availability from renewable resources andtheir biodegradability. Citrates can also be used in granularcompositions, especially in combination with zeolite and/or layeredsilicate builders. Oxydisuccinates are also especially useful in suchcompositions and combinations.

[0123] Also suitable in the detergent compositions of the presentinvention are the 3,3-dicarboxy-4-oxa-1,6-hexanedioates and the relatedcompounds disclosed in U.S. Pat. No. 4,566,984 to Bush, issued Jan. 28,1986. Useful succinic acid builders include the C5-C20 alkyl and alkenylsuccinic acids and salts thereof. A particularly preferred compound ofthis type is dodecenylsuccinic acid. Specific examples of succinatebuilders include: laurylsuccinate, myristylsuccinate, palmitylsuccinate,2-dodecenylsuccinate (preferred), 2pentadecenylsuccinate, and the like.Laurylsuccinates are the preferred builders of this group, and aredescribed in European Patent Application 200,263 to Barrat et al.,published Nov. 5, 1986.

[0124] Other suitable polycarboxylates are disclosed in U.S. Pat. No.4,144,226, Crutchfield et al, issued Mar. 13, 1979 and in U.S. Pat. No.3,308,067, Diehl, issued Mar. 7, 1967. See also U.S. Pat. No. 3,723,322to Diehl, issued Mar. 27, 1973.

[0125] Fatty acids, e.g., C12-C18 monocarboxylic acids, can also beincorporated into the compositions alone, or in combination with theaforesaid builders, especially citrate and/or the succinate builders, toprovide additional builder activity. Such use of fatty acids willgenerally result in a diminution of sudsing, which should be taken intoaccount by the formulator.

[0126] In situations where phosphorus-based builders can be used, andespecially in the formulation of bars used for hand-launderingoperations, the various alkali metal phosphates such as the well-knownsodium tripolyphosphates, sodium pyrophosphate and sodium orthophosphatecan be used. Phosphonate builders such asethane-1-hydroxy-1,1-diphosphonate and other known phosphonates (see,for example, U.S. Pat. No. 3,159,581 to Diehl, issued Dec. 1, 1964; U.S.Pat. No. 3,213,030 to Diehl, issued Oct. 19, 1965; U.S. Pat. No.3,400,148 to Quimby, issued Sep. 3, 1968; U.S. Pat. No. 3,422,021 toRoy, issued Jan. 14, 1969; and U.S. Pat. No. 3,422,137 to Quimby, issuedJan. 4, 1969) can also be used. Additional components which may be usedin the laundry detergent compositions of the present invention include,but are not limited to: alkoxylated polycarboxylates (to provide, e.g.,additional grease stain removal performance), bleaching agents, bleachactivators, bleach catalysts, brighteners, chelating agents, clay soilremoval/anti-redeposition agents, dye transfer inhibiting agents,additional enzymes (including lipases, amylases, hydrolases, and otherproteases), fabric softeners, polymeric soil release agents, polymericdispersing agents, and suds suppressors. The compositions herein mayfurther include one or more other detergent adjunct materials or othermaterials for assisting or enhancing cleaning performance, treatment ofthe substrate to be cleaned, or to modify the aesthetics of thedetergent composition (e.g., perfumes, colorants, dyes, etc.). Thedetergent compositions herein may further comprise other known detergentcleaning components including alkoxylated polycarboxylates, bleachingcompounds, brighteners, chelating agents, clay soilremoval/antiredeposition agents, dye transfer inhibiting agents,enzymes, enzyme stabilizing systems, fabric softeners, polymeric soilrelease agents, polymeric dispersing agents, suds suppressors. Thedetergent composition may also comprise other ingredients includingcarriers, hydrotropes, processing aids, dyes or pigments, solvents forliquid formulations, solid fillers for bar compositions.

[0127] Method of Treating or Preventing Skin Flaking

[0128] Another aspect of the present invention relates to a method oftreating or preventing skin flaking. The method comprises topicalapplication of a safe and effective amount of a composition comprisingthe protease EOS.

[0129] Herein, “safe and effective amount” means an amount of proteaseEOS high enough to provide a significant positive modification of thecondition to be treated, but low enough to avoid serious side effects(at a reasonable benefit/risk ratio), within the scope of sound medicaljudgment. A safe and effective amount of protease EOS will vary with theparticular condition being treated, the age and physical condition ofthe subject being treated, the severity of the condition, the durationof the treatment, the nature of concurrent therapy and like factors.

[0130] Suitable compositions for use in the subject method include theabove-described skin care compositions, including hair care compositions(for example, treating/preventing dandruff caused by skin flaking.

[0131] The following examples illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1 Plasmid Manipulations

[0132] All molecular biological methods were in accordance with thosepreviously described (Maniatis et al. (1989). 1-1626). Oligonucleotideswere purchased from Ransom Hill Biosciences (Ransom Hill, Calif.) andall restriction endonucleases and other DNA modifying enzymes were fromNew England Biolabs (Beverly, Mass.) unless otherwise specified. Theprotease EOS expression construct was made in the baculovirus expressionvector pFastBac1 (Life Technologies, Gaithersberg, Md.) as describedbelow. All construct manipulations were confirmed by dye terminatorcycle sequencing using Allied Biosystems 377 fluorescent sequencers(Perkin Elmer, Foster City, Calif.).

[0133] Acquisition of Protease EOS cDNA

[0134] Eosinophil RNA was isolated from pooled diseased eosinophilsobtained from allergic asthmatic individuals. Eosinophils were lysedimmediately following collection and purification in a buffer containingguanidinium isothiocyanate. The lysate was spun through CsCl to obtaintotal RNA, followed by poly A isolation using oligotex latex beads. ThecDNA synthesis was initiated using a NotI-oligo(dT) primer anddouble-stranded cDNA was blunted, ligated to Sal I adaptors, digestedwith Not I, size-selected, and cloned into the Not I and Sal I sites ofthe pSPORT1 vector (Life Technologies, Gaithersberg, Md.). A clone,corresponding to the full-length protease EOS cDNA, contained an openreading frame of 855 nucleotides (FIG. 1), and had homology to other S1serine proteases. This clone is also likely to contain the entire 3′untranslated since an AATAAA motif resides 18 nucleotides upstream of apoly A stretch of 50 nucleotides (in this particular clone, data notshown) a subset of which is presented in FIG. 1. Homology searches ofthe Genbank database with the protease EOS cDNA indicated that this wasa novel cDNA but had identity with the human cosmid clone (407D8,Genbank accession # AC005570), which maps to chromosome 16p13.3,indicating the position of protease EOS gene. The deduced open readingframe encodes a preproEOS protein of 284 amino acids (FIG. 1) , with anestimated molecular mass (M_(r)) of about 30-Kd, and a strong homologyto other serine proteases. The catalytic triad residues H, D and S arelocated at positions 77, 126 and 231, respectively. The zymogenactivation sequence MSSR-IVGG (positions 33 to 40) is very similar tothat of other S1 serine proteases and predicts a mature protein of 284amino acids. A signal peptide of 22 amino acids is predicted bystatistical method (Von Heijne (1986). Nucleic Acids Res. 14:4683-90)indicating a pro peptide of 14 amino acids. FASTA homology against theSWISS-PROT database indicates that the top match to protease EOS is thehuman prostasin precursor EC 3.4.21 SW:Q16651 (Yu et al. (1995). J.Biol. Chem. 270:13483-9; Yu et al. (1996). Genomics 32:334-40) (47.1%identity in 276 aa. overlap). The next match is dog tryptase precursorEC 3.4.21.59 SW:P15944 (Vanderslice et al. (1989). Biochemistry28:4148-55) (48.2% identity in 274 aa. overlap) . The next matchcorresponding to a known protein is human beta-tryptase precursor;tryptase 2 EC 3.4.21.59 SW:P20231 (Miller et al. (1990). J. Clin.Invest. 86:864-700) (44.3% identity in 273 aa. overlap). A phylogenetictree of an alignment of the deduced protease EOS amino acid sequencewith other members of the S1 serine protease family is shown in FIG. 2as determined using the MegAlign 3.1.7 program (DNASTAR Inc., Madison,Wis.).

EXAMPLE 2 Tissue Distribution of the Protease EOS mRNA

[0135] We employed a highly sensitive PCR profiling technique toidentify the tissue distribution of protease EOS mRNA. For thisapplication, several human cDNA libraries (all were from Clontech, (PaloAlto, Calif.) except the CHRF-288 megakaryocytic cell line and human gelfiltered platelet libraries which we constructed using the ZAP ExpresscDNA system (Stratagene, La Jolla, Calif.). The PCR primers for theprofiling analysis were as follows: SEQ.ID.NO.:2:5′-GAGAAAGTCAGATTCACAGC-3′ SEQ.ID.NO.:3: 5′-CTGCTTAGGGTCTCTTTAGG-3′

[0136] Briefly, the 50□l PCR reactions used 1□l of diluted phage stock(−10⁸ to 10¹⁰ pfu/ml) from each of the cDNA libraries tested. Reactionswere initially denatured at 94° C. for 5 minutes and subjected to 35cycles of 94° C. for 20 seconds; 56° C. for 20 seconds; and then 72° C.for 30 seconds followed by a final 72° C. elongation for 10 minutes Anested primer probe of the sequence SEQ.ID.NO.:4:5′-TGAGCGGCCTTTAAGAGTTGAGAGACAGCCGGCAGGGAAT-3 was radiolabeled usinggamma ³²P-ATP and T4 polynucleotide kinase (Life Technologies,Gaithersberg, Md.) and unincorporated label was removed, following thereaction, using a QIAquick nucleotide removal column (Qiagen, Valencia,Calif.). The ³²P end-labeled nested primer probe (1×10⁵ cpm) wascombined with 10□l of each sample following the PCR reaction. The PCRproduct-probe mixtures were denatured at 94° C. for 5 minutes;hybridized at 60° C. for 15 minutes, and cooled to 4° C. The annealedsamples (10□l) were electrophoresed in 6% Tris-Borate-EDTAnon-denaturing polyacrylamide gels (Novex) , dried and exposed byautoradiography. A PCR profile of the cDNA libraries used in FIG. 3 withbeta-actin PCR primers and labeled nested primer probe produced abeta-actin PCR product in all samples examined.

[0137] As seen in FIG. 3, the distribution of protease EOS mRNA ishighly restricted to specific tissues and cell types. The tissue typesexpressing the protease EOS transcript are retina, ovary, and spleen,stomach, and to a lesser extent thymus, uterus and thalamus. Ofparticular significance is that EOS protease mRNA is not expressed inpancreas, liver or prostate, tissues normally found to express numerousserine protease genes. It is of interest that we detect the protease EOSPCR product in cDNA libraries constructed from human gel filteredplatelets. This strongly suggests that protease EOS is expressed inhuman platelets. The platelets used to construct the cDNA libraryanalyzed in the PCR tissue profile of FIG. 3 contained extremely lowlevels of contaminating erythrocytes and other blood cells (<1 per 10⁶platelets), so we can not rule out the possibility of leukocytecontamination, which would generate a false positive signal in thissensitive PCR assay. Cell localization by in situ hybridization orsensitive in situ PCR, and confirmation with protease EOS specificantisera, could be employed to address this issue.

EXAMPLE 3 Construct Generation for the Expression of Active Protease EOS

[0138] Since members of the S1 protease family are most oftensynthesized as inactive zymogen precursors, and require limitedproteolysis to become proteolytically active, we have developed azymogen activation construct to express and permit the genericactivation of heterologous serine protease cDNAs. This constructfeatures a bovine preprolactin signal sequence fused in-frame with theMoAb M2 anti-FLAG antibody epitope as previously described (Ishii et al.(1993). J. Biol. Chem. 268:9780-6) for the purposes of secretion andantibody detection respectively (PF). Significantly, this construct alsocontains the enterokinase cleavage site from human trypsinogen I (EK)fused in-frame and downstream from the signal sequence. At theC-terminus, preceding a stop codon, are additional sequences encodingthe hemagglutinin (HA) epitope and 6 histidine (6XHIS) codons fordetection with the anti-HA antibody MoAb 12 CA5 (Boehringer MannheimCorp., Indianapolis, Ind.) and affinity purification on nickel resinsrespectively. A unique Xba I restriction enzyme site, immediatelyupstream of the epitope/affinity tag sequence and downstream of the PFEKprepro sequence described above, and is the point of in-frame insertionof the catalytic domain of a heterologous serine protease cDNA (FIG. 4).The zymogen activation vector described above has been cloned into amodified pFastBacl transplacement plasmid to generate PFEK-HA6XHIS-TAGFB.

[0139] The purified plasmid DNA of the full length protease EOS cDNA wasused as a template in a 100 □l preparative PCR reaction using theAdvantage-GC cDNA Polymerase Mix (Clontech, Palo Alto, Calif.) inaccordance with the manufacturer's recommendations. The primers usedSEQ.ID.NO.:5: EOS Xba-U 5′-GGGATCTAGAGGACGGAGAGTGGCCGTGGC-3′SEQ.ID.NO.:7: EOS Xba-L 5′-CTCATCTAGAAGCATTAGAAGTGACGCGAGCCTG-3′contained Xba I cleavable ends, and were designed to flank the catalyticdomain of the protease EOS and generate the protease EOS Xba I catalyticcassette. The preparative PCR reaction was run at 18 cycles of 94° C.for 30 seconds ; 68° C. for 2.5 minutes.

[0140] The preparative PCR product was phenol/CHCl₃ (1:1) extractedonce, CHCl₃ extracted, and then EtOH precipitated with glycogen(Boehringer Mannheim Corp., Indianapolis, Ind.) and carrier. Theprecipitated pellet was rinsed with 70% EtOH, dried by vacuum, andresuspended in 80 ul H₂0, 10 ul restriction buffer number 2 and 1 ul100× BSA (New England Biolabs, Beverly, Mass.). The product was digestedfor 3 hr. at 37° C. with 200 units Xba I restriction enzyme (New EnglandBiolabs, Beverly, Mass.). The Xba I digested product was phenol/CHCl₃(1:1) extracted once, CHCl₃ extracted, EtOH precipitated, rinsed with70% EtOH, and dried by vacuum. For purification from contaminatingtemplate plasmid DNA, the product was electrophoresed through 1.0% lowmelting temperature agarose (Life Technologies, Gaithersberg, Md.) gelsin TAE buffer (40 mM Tris-Acetate, 1 mM EDTA pH 8.3) and excised fromthe gel. An aliquot of the excised product was then used for in-gelligations with the Xba I digested, dephosphorylated and gel purified,zymogen activation vector described above. Clones containing the EOS Xbacassette, inserted in the correct orientation to generate the constructPFEK-protease EOS-HA6XHIS-TAG 64, were confirmed by sequence analyses toensure that the proper translational register with respect to theNH₂-terminal PFEK prepro sequence and C-terminal HA6XHISepitope/affinity tag was maintained.

EXAMPLE 4 Expression of Recombinant Protease EOS

[0141] The recombinant bacmid containing the PFEK-protease EOS-HA6XHISconstruct was prepared from bacterial transformation, selection, growth,purification and PCR confirmation in accordance with the manufacturer'srecommendations. Cultured Sf9 insect cells (ATCC CRL-1711) weretransfected with purified bacmid DNA and several days later, conditionedmedia containing recombinant PFEK-EOS-HA6×XHIS baculovirus was collectedfor viral stock amplification. Sf9 cells growing in Sf-900 II SFM at adensity of 2×10⁶/ml were infected at a multiplicity of infection of 2 at27° C. for 80 hours, and cell pellets were collected for purification ofPFEK-EOS-HA6XHIS.

EXAMPLE 5 Purification, and Activation of Recombinant Protease EOS

[0142] Cells were lyzed on ice in 20 mM Tris (pH7.4), 150 mM NaCl, 1%Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM PMSF, leupeptin (1 □g/ml), andpepstatin (1 □g/ml). Cell lysates were mixed with anti-FLAG M2 affinitygel (Eastman Kodak Co., New Haven, Conn.) and bound at 4° C. for 3 hourswith gentle rotation. The zymogen-bound resin was washed 3 times withTBS buffer (50 mM Tris-HCl, 150 mM NaCl at a final pH of 7.5) , andeluted by competition with FLAG peptide (100 □g/ml) in TBS buffer. Theeluted zymogen was dialyzed overnight against TBS in Spectra/Pormembrane (MWCO: 12,000-14,000) (Spectra Medical Industries, Inc.,Huston, Tex.). Ni-NTA (150 □l of a 50% slurry/per 100 □g of zymogen)(Qiagen, Valencia, Calif.) was added to 5 ml the dialyzed sample andmixed by shaking at 4° C. for 60 minutes The zymogen-bound resin waswashed 3 times with wash buffer [10 mM Tris-HCl (pH 8.0), 300 mM NaCl,and 15 mM imidazole], followed by with a 1.5 ml wash with ds H₂O.Zymogen cleavage was carried out by adding enterokinase (10 U per 50 □gof zymogen) (Novagen, Inc., Madison Wis.; or Sigma, St. Louis, Mo.) tothe zymogen-bound Ni-NTA beads in a small volume at room temperatureovernight with gentle shaking in a buffer containing 20 mM Tris-HCl (pH7.4), 50 mM NaCl, and 2.0 mM CaCl₂. The resin was then washed twice with1.5 ml wash buffer. The activated protease EOS-HA6XHIS was eluted withelution buffer [20 mM Tris-HCl (pH 7.8), 250 mM NaCl, and 250 mMimidazole] . Eluted protein concentration was determined by a Micro BCAKit (Pierce, Rockford, Ill.) using bovine serum albumin as a standard.Amidolytic activities of the activated protease EOS-HA6XHIS wasmonitored by release of para-nitroaniline (pNA) from the syntheticsubstrates indicated in FIG. 6. The chromogenic substrates used in thesestudies were all commercially available (Bachem California Inc.,Torrance, Pa.; American Diagnostica Inc., Greenwich, Conn.; KabiPharmacia Hepar Inc., Franklin, Ohio). Assay mixtures containedchromogenic substrates at 500 uM and 10 mM Tris-HCl (pH 7.8), 25 mMNaCl, and 25 mM imidazole. Release of pNA was measured over 120 minutesat 37° C. on a micro-plate reader (Molecular Devices, Menlo Park,Calif.) with a 405 nm absorbance filter. The initial reaction rates(Vmax, mOD/min) were determined from plots of absorbance versus timeusing Softmax (Molecular Devices, Menlo Park, Calif.). The specificactivities (nmole pNA produced /min/ug protein) of the activatedprotease EOS-HA6XHIS for the various substrates are presented in FIG. 6.No measurable chromogenic amidolytic activity was detected with thepurified unactivated PFEK-protease EOS-HA6XHIS zymogen.

[0143] Electrophoresis and Western Blotting Detection of RecombinantProteases EOS

[0144] Samples of the purified PFEK-protease EOS-HA6XHIS zymogen oractivated protease EOS-HA6XHIS, denatured in the presence of thereducing agent dithiothreitol (DTT), were analyzed by SDS-PAGE (Bio Rad,Hercules Calif.) stained with Coomassie Brilliant Blue. For Westernblotting, gels were electrotransfer to Hybond ECL membranes (Amersham,Arlington Heights, Ill.). The FLAG-tagged PFEK-protease EOS-HA6XHISzymogen expressed from infected Sf9 cells was detected with anti-Flag M2antibody (Eastman Kodak Co., New Haven, Conn.). The secondary antibodywas a goat-anti-mouse IgG (H+L), horseradish peroxidase-linked F(ab′)2fragment, (Boehringer Mannheim Corp., Indianapolis, Ind.) and wasdetected by the ECL kit (Amersham, Arlington Heights, Ill.).

EXAMPLE 6 Chromogenic Assay

[0145] Amidolytic activities of the activated serine proteases aremonitored by release of para-nitroaniline (pNA) from syntheticsubstrates that are commercially available (Bachem California Inc.,Torrance, Pa.; American Diagnostica Inc., Greenwich, Conn.; KabiPharmacia Hepar Inc., Franklin, Ohio). Assay mixtures containchromogenic substrates in 500 uM and 10 mM TRIS-HCl (pH 7.8), 25 mMNaCl, and 25 mM imidazole. Release of pNA is measured over 120 min at37° C. on a micro-plate reader (Molecular Devices, Menlo Park, Calif.)with a 405 nm absorbance filter. The initial reaction rates (Vmax,mOD/min) are determined from plots of absorbance versus time usingSoftmax (Molecular Devices, Menlo Park, Calif.) . Compounds thatmodulate a serine protease of the present invention are identifiedthrough screening for the acceleration, or more commonly, the inhibitionof the proteolytic activity. Although in the present case chromogenicactivity is monitored by an increase in absorbance, fluorogenic assaysor other methods such as FRET to measure proteolytic activity asmentioned above, can be employed. Compounds are dissolved in anappropriate solvent, such as DMF, DMSO, methanol, and diluted in waterto a range of concentrations usually not exceeding 100 uM and aretypically tested, though not limited to, a concentration of 1000-foldthe concentration of protease. The compounds are then mixed with theprotein stock solution, prior to addition to the reaction mixture.Alternatively, the protein and compound solutions may be addedindependently to the reaction mixture, with the compound being addedeither prior to, or immediately after, the addition of the proteaseprotein

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1 9 1 1613 DNA Homo sapiens 1 ccacgcgtcc gaccagagtc caagccctaggcagtgccac ccttacccag cccagccttg 60 aagacagaat gagaggggtt tcctgtctccaggtcctgct ccttctggtg ctgggagctg 120 ctgggactca gggaaggaag tctgcagcctgcgggcagcc ccgcatgtcc agtcggatcg 180 ttgggggccg ggatggccgg gacggagagtggccgtggca ggcgagcatc cagcatcctg 240 gggcacacgt gtgcgggggg tcgctcatcgccccccagtg ggtgctgaca gcggcgcact 300 gcttccccag gagggcactg ccagctgagtaccgcgtgcg cctgggggcg ctgcgtctgg 360 gctccacctc gccccgcacg ctctcggtgcccgtgcgacg ggtgctgctg cccccggact 420 actccgagga cggggcccgc ggcgacctggcactgctgca gctgcgtcgc ccggtgcccc 480 tgagcgctcg cgtccaaccc gtctgcctgcccgtgcccgg cgcccgcccg ccgcccggca 540 caccatgccg ggtcaccggc tggggcagcctccgcccagg agtgcccctc ccagagtggc 600 gaccgctaca aggagtaagg gtgccgctgctggactcgcg cacctgcgac ggcctctacc 660 acgtgggcgc ggacgtgccc caggctgagcgcattgtgct gcctgggagt ctgtgtgccg 720 gctaccccca gggccacaag gacgcctgccagggtgattc tgggggacct ctgacctgcc 780 tgcagtctgg gagctgggtc ctggtgggcgtggtgagctg gggcaagggt tgtgccctgc 840 ccaaccgtcc aggggtctac accagtgtggccacatatag cccctggatt caggctcgcg 900 tcacttctaa tgctagccgg tgaggctgacctggagccag ctgctggggt ccctcagcct 960 cctggttcat ccaggcacct gcctataccccacatccctt ctgcctcgag gccaagatgc 1020 ctaaaaaagc taaaggccac cccaccccccacccaccacc ttctggctcc tctcctcttt 1080 ggggatcacc agctctgact ccaccaaccctcatccagga atctgccatg agtcccaggg 1140 agtcacactc cccactccct tcctggcttgtatttacttt tcttggccct ggccagggct 1200 gggcgcaagg cacgcagtga tgggcaaaccaattgctgcc catctggcct gtgtgcccat 1260 ctttttctgg agaaagtcag attcacagcatgacagagat ttgacaccag ggagatcctc 1320 catagctggc tttgaggaca cggggaccacagccatgagc ggcctctaag agctgagaga 1380 cagccggcag ggaatcggaa ccctcagacccacagccgca aggcactgga ttctggcagc 1440 accctgaagg agctgggaag taagttcttccccagcctcc agataagagc cccgccggcc 1500 aatcccttca tttcaaccta aagagaccctaagcagagaa cctagctgag ccactcctga 1560 cctacaaagt tgtgacttaa taaatgtgtgctttaagctg ccaaaaaaaa aaa 1613 2 20 DNA Artificial Sequence Descriptionof Artificial Sequence oligonucleotide 2 gagaaagtca gattcacagc 20 3 20DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide 3 ctgcttaggg tctctttagg 20 4 40 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 4 tgagcggcctttaagagttg agagacagcc ggcagggaat 40 5 30 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide 5 gggatctagaggacggagag tggccgtggc 30 6 34 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide 6 ctcatctaga agcattagaa gtgacgcgagcctg 34 7 284 PRT Homo sapiens 7 Met Arg Gly Val Ser Cys Leu Gln Val LeuLeu Leu Leu Val Leu Gly 1 5 10 15 Ala Ala Gly Thr Gln Gly Arg Lys SerAla Ala Cys Gly Gln Pro Arg 20 25 30 Met Ser Ser Arg Ile Val Gly Gly ArgAsp Gly Arg Asp Gly Glu Trp 35 40 45 Pro Trp Gln Ala Ser Ile Gln His ProGly Ala His Val Cys Gly Gly 50 55 60 Ser Leu Ile Ala Pro Gln Trp Val LeuThr Ala Ala His Cys Phe Pro 65 70 75 80 Arg Arg Ala Leu Pro Ala Glu TyrArg Val Arg Leu Gly Ala Leu Arg 85 90 95 Leu Gly Ser Thr Ser Pro Arg ThrLeu Ser Val Pro Val Arg Arg Val 100 105 110 Leu Leu Pro Pro Asp Tyr SerGlu Asp Gly Ala Arg Gly Asp Leu Ala 115 120 125 Leu Leu Gln Leu Arg ArgPro Val Pro Leu Ser Ala Arg Val Gln Pro 130 135 140 Val Cys Leu Pro ValPro Gly Ala Arg Pro Pro Pro Gly Thr Pro Cys 145 150 155 160 Arg Val ThrGly Trp Gly Ser Leu Arg Pro Gly Val Pro Leu Pro Glu 165 170 175 Trp ArgPro Leu Gln Gly Val Arg Val Pro Leu Leu Asp Ser Arg Thr 180 185 190 CysAsp Gly Leu Tyr His Val Gly Ala Asp Val Pro Gln Ala Glu Arg 195 200 205Ile Val Leu Pro Gly Ser Leu Cys Ala Gly Tyr Pro Gln Gly His Lys 210 215220 Asp Ala Cys Gln Gly Asp Ser Gly Gly Pro Leu Thr Cys Leu Gln Ser 225230 235 240 Gly Ser Trp Val Leu Val Gly Val Val Ser Trp Gly Lys Gly CysAla 245 250 255 Leu Pro Asn Arg Pro Gly Val Tyr Thr Ser Val Ala Thr TyrSer Pro 260 265 270 Trp Ile Gln Ala Arg Val Thr Ser Asn Ala Ser Arg 275280 8 1130 DNA Artificial Sequence Description of Artificial SequenceNucleic acid sequence of EOS zymogen fusion gene 8 gaattcacca ccatggacagcaaaggttcg tcgcagaaat cccgcctgct cctgctgctg 60 gtggtgtcaa atctactcttgtgccagggt gtggtctccg actacaagga cgacgacgac 120 gtggacgcgg ccgctcttgctgcccccttt gatgatgatg acaagatcgt tgggggctat 180 gctctagagg acggagagtggccgtggcag gcgagcatcc agcatcctgg ggcacacgtg 240 tgcggggggt cgctcatcgccccccagtgg gtgctgacag cggcgcactg cttccccagg 300 agggcactgc cagctgagtaccgcgtgcgc ctgggggcgc tgcgtctggg ctccacctcg 360 ccccgcacgc tctcggtgcccgtgcgacgg gtgctgctgc ccccggacta ctccgaggac 420 ggggcccgcg gcgacctggcactgctgcag ctgcgtcgcc cggtgcccct gagcgctcgc 480 gtccaacccg tctgcctgcccgtgcccggc gcccgcccgc cgcccggcac accatgccgg 540 gtcaccggct ggggcagcctccgcccagga gtgcccctcc cagagtggcg accgctacaa 600 ggagtaaggg tgccgctgctggactcgcgc acctgcgacg gcctctacca cgtgggcgcg 660 gacgtgcccc aggctgagcgcattgtgctg cctgggagtc tgtgtgccgg ctacccccag 720 ggccacaagg acgcctgccagggtgattct gggggacctc tgacctgcct gcagtctggg 780 agctgggtcc tggtgggcgtggtgagctgg ggcaagggtt gtgccctgcc caaccgtcca 840 ggggtctaca ccagtgtggccacatatagc ccctggattc aggctcgcgt cacttctaat 900 gcttctagat acccctacgatgtgcccgat tacgccgcta gacatcacca tcaccatcac 960 tagcggccgc ttccctttagtgagggttaa tgcttcgagc agacatgata agatacattg 1020 atgagtttgg acaaaccacaactagaatgc agtgaaaaaa atgctttatt tgtgaaattt 1080 gtgatgctat tgctttatttgtaaccatta taagctgcaa taaacaagtt 1130 9 316 PRT Artificial SequenceDescription of Artificial Sequence Amino acid sequence of EOS zymogenfusion gene 9 Met Asp Ser Lys Gly Ser Ser Gln Lys Ser Arg Leu Leu LeuLeu Leu 1 5 10 15 Val Val Ser Asn Leu Leu Leu Cys Gln Gly Val Val SerAsp Tyr Lys 20 25 30 Asp Asp Asp Asp Val Asp Ala Ala Ala Leu Ala Ala ProPhe Asp Asp 35 40 45 Asp Asp Lys Ile Val Gly Gly Tyr Ala Leu Glu Asp GlyGlu Trp Pro 50 55 60 Trp Gln Ala Ser Ile Gln His Pro Gly Ala His Val CysGly Gly Ser 65 70 75 80 Leu Ile Ala Pro Gln Trp Val Leu Thr Ala Ala HisCys Phe Pro Arg 85 90 95 Arg Ala Leu Pro Ala Glu Tyr Arg Val Arg Leu GlyAla Leu Arg Leu 100 105 110 Gly Ser Thr Ser Pro Arg Thr Leu Ser Val ProVal Arg Arg Val Leu 115 120 125 Leu Pro Pro Asp Tyr Ser Glu Asp Gly AlaArg Gly Asp Leu Ala Leu 130 135 140 Leu Gln Leu Arg Arg Pro Val Pro LeuSer Ala Arg Val Gln Pro Val 145 150 155 160 Cys Leu Pro Val Pro Gly AlaArg Pro Pro Pro Gly Thr Pro Cys Arg 165 170 175 Val Thr Gly Trp Gly SerLeu Arg Pro Gly Val Pro Leu Pro Glu Trp 180 185 190 Arg Pro Leu Gln GlyVal Arg Val Pro Leu Leu Asp Ser Arg Thr Cys 195 200 205 Asp Gly Leu TyrHis Val Gly Ala Asp Val Pro Gln Ala Glu Arg Ile 210 215 220 Val Leu ProGly Ser Leu Cys Ala Gly Tyr Pro Gln Gly His Lys Asp 225 230 235 240 AlaCys Gln Gly Asp Ser Gly Gly Pro Leu Thr Cys Leu Gln Ser Gly 245 250 255Ser Trp Val Leu Val Gly Val Val Ser Trp Gly Lys Gly Cys Ala Leu 260 265270 Pro Asn Arg Pro Gly Val Tyr Thr Ser Val Ala Thr Tyr Ser Pro Trp 275280 285 Ile Gln Ala Arg Val Thr Ser Asn Ala Ser Arg Tyr Pro Tyr Asp Val290 295 300 Pro Asp Tyr Ala Ala Arg His His His His His His 305 310 315

What is claimed is:
 1. An isolated and purified nucleic acid moleculethat encodes protease EOS, and functional derivatives thereof.
 2. Theisolated and purified nucleic acid molecule of claim 1, having anucleotide sequence selected from a group consisting of: (SEQ.ID.NO.:1),(SEQ. ID.NO.:8) and functional derivatives thereof.
 3. The isolated andpurified nucleic acid molecule of claim 1, wherein said nucleic acidmolecule is selected from a group consisting of cDNA, RNA, and genomicDNA.
 4. An expression vector for expression of protease EOS protein in arecombinant host, wherein said vector contains a nucleic acid sequenceencoding protease EOS protein, and functional derivatives thereof. 5.The expression vector of claim 4, wherein the nucleic acid sequenceencoding protease EOS protein is selected from a group consisting of(SEQ. ID.NO.:1) , (SEQ.ID.NO.:8), nucleotide sequence encoding theprotein of SEQ.ID.NO.:7, and functional derivatives thereof.
 6. Theexpression vector of claim 4, wherein said nucleic acid molecule isselected from a group consisting of cDNA, RNA, nucleic acid sequenceencoding SEQ.ID.NO.:7, and genomic DNA.
 7. A recombinant host cellcontaining the expression vector of claim
 4. 8. The recombinant hostcell of claim 7, wherein said expression vector contains a nucleotidesequence selected from a group consisting of (SEQ.ID.NO.:1),(SEQ.ID.NO.:8), nucleic acid sequence encoding SEQ.ID.NO.:7, andfunctional derivatives thereof.
 9. The recombinant host cell of claim 7,wherein said nucleotide sequence is selected from a group consisting ofcDNA, RNA, nucleic acid sequence encoding SEQ.ID.NO.:7, and genomic DNA.10. A protein in substantially pure form that functions as protease EOSprotein.
 11. The protein according to claim 10, having an amino acidsequence selected from a group consisting of (SEQ.ID.NO.:7),(SEQ.ID.NO.:9) and functional derivatives thereof.
 12. A monospecificantibody immunologically reactive with protease EOS protein.
 13. Theantibody of claim 12, wherein the antibody blocks protease activity ofthe protein.
 14. A process for expression of protease EOS protein in arecombinant host cell, comprising: (a) transferring the expressionvector of claim 4 into suitable host cells; and (b) culturing the hostcells of step (a) under conditions which allow expression of theprotease EOS protein from the expression vector.
 15. A method ofidentifying compounds that modulate protease EOS protein activity,comprising: (a) combining a modulator of protease EOS protein activity,protease EOS protein, and a labeled substrate; and (b) measuring achange in the labeled substrate.
 16. The method of claim 15 wherein thelabeled substrate is selected from the group consisting of fluorogenic,colormetric, radiometric, and fluorescent resonance energy transfer(FRET)
 17. A compound active in the method of claim 15, wherein saidcompound is a modulator of protease EOS serine protease activity. 18.The compound of claim 17, wherein said compound is an agonist orantagonist of protease EOS serine protease activity.
 19. The compound ofclaim 17, wherein said compound is a modulator of expression of proteaseEOS serine protein.
 20. A method of treating a patient in need of suchtreatment for a condition that is mediated by protease EOS, comprisingadministration of the compound of claim
 17. 21. A kit comprising thenucleic acid sequence selected from the group consisting of SEQ.ID.NO.:1and SEQ.ID.NO.:8, nucleotide sequence encoding SEQ.ID.NO.:7, andfragments thereof.
 22. A kit comprising the serine protease EOS proteinselected from the group consisting of SEQ.ID.NO.:7 and SEQ.ID.NO.:9, andfragments or derivatives thereof.
 23. A pharmaceutical compositioncomprising the protein of claim
 10. 24. The pharmaceutical compositionof claim 23 wherein said composition is a topical skin care composition.25. A non-pharmaceutical composition comprising the protein of claim 10.26. The non-pharmaceutical composition of claim 25 wherein theformulation is selected from the group consisting of laundry detergent,shampoo, hard surface cleaning compositions, and dishcare cleaningcomposition.
 27. A method of treating an imbalance of desquamationcomprising topical application of the composition of claim 24.